Seed specific USP promoters for expressing genes in plants

ABSTRACT

The present invention relates to the field of plant genetic engineering. More specifically, the present invention relates to seed specific gene expression. The present invention provides promoters capable of transcribing heterologous nucleic acid sequences in seeds, and methods of modifying, producing, and using the same.

This application claims the benefit of the filing date of theprovisional Application U.S. Ser. No. 60/377,236, filed May 3, 2002.

The present invention relates to the field of plant genetic engineering.More specifically, the present invention relates to seed specific geneexpression. The present invention provides promoters capable oftranscribing heterologous nucleic acid sequences in seeds, and methodsof modifying, producing, and using the same.

Seeds provide an important source of dietary protein for humans andlivestock. However, the protein content of seeds is often incomplete.For example, many seed proteins are deficient in one or more essentialamino acids. This deficiency may be overcome by genetically modifyingthe native or non-native proteins to have a more nutritionally completecomposition of amino acids (or some other desirable feature) and tooverexpress the modified proteins in the transgenic plants.Alternatively, one or more genes could be introduced into a crop plantto manipulate the metabolic pathways and modify the free amino acidcontent. These approaches are useful in producing crops exhibitingimportant agricultural (e.g., yield), nutritional, and pharmaceuticalproperties.

Despite the availability of many molecular tools, the geneticmodification of seeds is often constrained by an insufficientaccumulation of the engineered protein. Many intracellular processes mayimpact the overall protein accumulation, including transcription,translation, protein assembly and folding, transport, and proteolysis.Intervention in one or more of these processes can increase the amountof protein produced in genetically engineered seeds.

Introduction of a gene can cause deleterious effects on plant growth anddevelopment. Under such circumstances, the expression of the gene mayneed to be limited to the desired target tissue. For example, it mightbe necessary to express an amino acid deregulation gene in aseed-specific or seed-enhanced fashion to avoid an undesired phenotypethat may affect yield or other agronomic traits.

The promoter portion of a gene plays a central role in controlling geneexpression. Along the promoter region, the transcription machinery isassembled and transcription is initiated. This early step is often a keyregulatory step relative to subsequent stages of gene expression.Transcription initiation at the promoter may be regulated in severalways. For example, a promoter may be induced by the presence of aparticular compound, express a gene only in a specific tissue, orconstitutively express a coding sequence. Thus, transcription of acoding sequence may be modified by operably linking the coding sequenceto promoters with different regulatory characteristics.

SUMMARY OF THE INVENTION

The present invention includes and provides a transformed plantcontaining a nucleic acid molecule that comprises a promoter comprisinga nucleic acid sequence that hybridizes under stringent conditions witha nucleic acid sequence selected from the group consisting of SEQ IDNOS: 1, 2, 3, 4, 9, 10, and 11, and complements thereof. The presentinvention includes and provides a transformed plant containing a nucleicacid molecule that comprises a promoter comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4,9, 10, and 11, and complements thereof operably linked to a structuralnucleic acid sequence.

The present invention includes and provides a transformed plantcontaining a nucleic acid molecule that comprises a promoter comprisinga nucleic acid sequence selected from the group consisting of: a nucleicacid sequence having greater than about 85.5% identity to SEQ ID NO: 1or its complement, a nucleic acid sequence having greater than about85.5% identity to SEQ ID NO: 2 or its complement, a nucleic acidsequence having greater than about 97.1% identity to SEQ ID NO: 3 or itscomplement, and a nucleic acid sequence having greater than about 96.4%identity to SEQ ID NO: 4 or its complement.

The present invention includes and provides a method of producing atransformed plant comprising: providing a nucleic acid molecule thatcomprises a promoter comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11, andcomplements thereof, operably linked to a structural nucleic acidsequence; and transforming a plant with said nucleic acid molecule.

The present invention includes and provides a method of expressing astructural nucleic acid molecule in a seed comprising: growing atransformed plant containing a nucleic acid molecule that comprises apromoter comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11, and complementsthereof, operably linked to said structural nucleic acid molecule,wherein said transformed plant produces said seed and said structuralnucleic acid molecule is transcribed in said seed; and isolating saidseed.

The present invention includes and provides a method of obtaining a seedenhanced in a product of a structural nucleic acid molecule comprising:growing a transformed plant containing a nucleic acid molecule thatcomprises a promoter comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11, andcomplements thereof, operably linked to said structural nucleic acidmolecule, wherein said transformed plant produces said seed and saidstructural nucleic acid molecule is transcribed in said seed; andisolating said seed from said transformed plant.

The present invention includes and provides a method of obtaining mealenhanced in a product of a structural nucleic acid molecule comprising:growing a transformed plant containing a nucleic acid molecule thatcomprises a promoter comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11, andcomplements thereof, operably linked to said structural nucleic acidmolecule, wherein said transformed plant produces a seed and saidstructural nucleic acid molecule is transcribed in said seed; andpreparing said meal comprising said transformed plant or part thereof.

The present invention includes and provides a method of obtainingfeedstock enhanced in a product of a structural nucleic acid moleculecomprising: growing a transformed plant containing a nucleic acidmolecule that comprises a promoter comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 9, 10, and11, and complements thereof, operably linked to said structural nucleicacid molecule, wherein said transformed plant produces a seed and saidstructural nucleic acid molecule is transcribed in said seed; andpreparing said feedstock comprising said transformed plant or partthereof.

The present invention includes and provides a method of obtaining oilenhanced in a product of a structural nucleic acid molecule comprising:growing a transformed plant containing a nucleic acid molecule thatcomprises a promoter comprising a nucleic acid sequence that hybridizesunder stringent conditions with a nucleic acid sequence selected fromthe group consisting of SEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11, andcomplements thereof, operably linked to said structural nucleic acidmolecule, wherein said transformed plant produces a seed and saidstructural nucleic acid molecule is transcribed in said seed; andisolating said oil.

The present invention includes and provides a cell containing a vectorcomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11, and complements thereof.

The present invention includes and provides oil produced from one ormore seeds of a transformed plant containing a nucleic acid moleculethat comprises a promoter comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11, andcomplements thereof.

The present invention includes and provides oil produced from one ormore seeds of a transformed plant containing a nucleic acid moleculethat comprises a promoter comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11, andcomplements thereof, operably linked to a structural nucleic acidsequence, wherein the promoter is heterologous with respect to thestructural nucleic acid sequence.

The present invention includes and provides a seed generated by atransformed plant containing a nucleic acid molecule that comprises: apromoter comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11, and complementsthereof.

The present invention includes and provides feedstock comprising atransformed plant or part thereof containing a nucleic acid moleculethat comprises a promoter comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11, andcomplements thereof.

The present invention includes and provides meal comprising plantmaterial from a transformed plant containing a nucleic acid moleculethat comprises a promoter comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11, andcomplements thereof.

The present invention includes and provides a container of seeds,wherein at least about 25% of said seeds comprise a promoter comprisinga nucleic acid sequence selected from the group consisting of SEQ IDNOS: 1, 2, 3, 4, 9, 10, and 11, and complements thereof, operably linkedto a structural nucleic acid sequence, wherein said promoter isheterologous with respect to the structural nucleic acid sequence.

The present invention includes and provides a substantially purifiednucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11, andcomplements thereof.

The present invention includes and provides a substantially purifiednucleic acid molecule having a nucleic acid sequence selected from thegroup consisting of: a nucleic acid sequence having greater than about85.5% identity to SEQ ID NO: 1 or its complement, a nucleic acidsequence having greater than about 85.5% identity to SEQ ID NO: 2 or itscomplement, a nucleic acid sequence having greater than about 97.1%identity to SEQ ID NO: 3 or its complement, and a nucleic acid sequencehaving greater than about 96.4% identity to SEQ ID NO: 4 or itscomplement.

The present invention includes and provides a transformed soybean plantcontaining a nucleic acid molecule that comprises a promoter comprisinga nucleic acid sequence that hybridizes under stringent conditions witha nucleic acid sequence selected from the group consisting of SEQ IDNOS: 1, 2, 3, 4, 5, 9, 10, and 11, and complements thereof.

The present invention includes and provides a transformed soybean plantcontaining a nucleic acid molecule that comprises a promoter comprisinga nucleic acid sequence selected from the group consisting of SEQ IDNOS: 1, 2, 3, 4, 5, 9, 10, and 11, and complements thereof operablylinked to a structural nucleic acid sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic of vector pMON13773.

FIG. 2 is a schematic of vector pMON58101.

FIG. 3 is a schematic of vector pMON58102.

FIG. 4 is a schematic of vector pMON58106.

FIG. 5 is a schematic of vector pMON58110.

FIG. 6 is a schematic of vector pMON58100.

FIG. 7 is a schematic of vector pMON58107.

FIG. 8 is a schematic of vector pMON58113.

FIG. 9 is a schematic of vector pMON55526.

FIG. 10 is a schematic of vector pMON58108.

FIG. 11 is a schematic of vector pMON39319.

FIG. 12 is a graph representing the relative GUS activity expressed intransgenic soybean under the control of multiple promoters.

FIG. 13 is a schematic of vector pMON58130.

FIG. 14 is a graph representing the relative GUS activity of variousconstructs.

FIG. 15 is a schematic of vector pMON63604.

FIG. 16 is a schematic of vector pMON63605.

FIG. 17 is a schematic of vector pMON55542.

FIG. 18 is a schematic of vector pMON63821.

FIG. 19 is a schematic of vector pMON63819.

FIG. 20 is a schematic of vector pMON63820.

FIG. 21 is a schematic of vector pMON63654.

BRIEF DESCRIPTION OF THE SEQUENCES

-   SEQ ID NO: 1 is a USP88 promoter sequence from Vicia faba.-   SEQ ID NO: 2 is an eUSP88 promoter sequence from Vicia faba.-   SEQ ID NO: 3 is a USP99 promoter sequence from Vicia faba.-   SEQ ID NO: 4 is a USP91 promoter sequence from Vicia faba.-   SEQ ID NO: 5 is a USP promoter sequence from Vicia faba.-   SEQ ID NO: 6 is a primer sequence for amplifying the USP promoter    from Vicia faba.-   SEQ ID NO: 7 is a primer sequence for amplifying the USP promoter    from Vicia faba.-   SEQ ID NO: 8 is a primer sequence for amplifying the USP promoter    from Vicia faba.-   SEQ ID NO: 9 is a USP99.5 promoter sequence from Vicia faba.-   SEQ ID NO: 10 is a USP95 promoter sequence from Vicia faba.-   SEQ ID NO: 11 is a USP68 promoter sequence from Vicia faba.-   SEQ ID NO: 12 is a primer sequence for amplifying a USP promoter    from Vicia faba.-   SEQ ID NO: 13 is a primer sequence for amplifying a USP promoter    from Vicia faba.-   SEQ ID NO: 14 is a primer sequence for amplifying a USP promoter    from Vicia faba.-   SEQ ID NO: 15 is a primer sequence for amplifying a USP promoter    from Vicia faba.-   SEQ ID NO: 16 is a primer sequence for amplifying a USP promoter    from Vicia faba.

Definitions

The following definitions are provided as an aid to understanding thedetailed description of the present invention.

The phrases “coding sequence,” “structural sequence,” and “structuralnucleic acid sequence” refer to a physical structure comprising anorderly arrangement of nucleotides. The nucleotides are arranged in aseries of triplets that each form a codon. Each codon encodes a specificamino acid. Thus, the coding sequence, structural sequence, andstructural nucleic acid sequence encode a series of amino acids forminga protein, polypeptide, or peptide sequence. The coding sequence,structural sequence, and structural nucleic acid sequence may becontained within a larger nucleic acid molecule, vector, or the like. Inaddition, the orderly arrangement of nucleotides in these sequences maybe depicted in the form of a sequence listing, figure, table, electronicmedium, or the like.

The phrases “DNA sequence,” “nucleic acid sequence,” and “nucleic acidmolecule” refer to a physical structure comprising an orderlyarrangement of nucleotides. The DNA sequence or nucleotide sequence maybe contained within a larger nucleotide molecule, vector, or the like.In addition, the orderly arrangement of nucleic acids in these sequencesmay be depicted in the form of a sequence listing, figure, table,electronic medium, or the like.

The term “expression” refers to the transcription of a gene to producethe corresponding mRNA and translation of this mRNA to produce thecorresponding gene product (i.e., a peptide, polypeptide, or protein).

The phrase “expression of antisense RNA” refers to the transcription ofa DNA to produce a first RNA molecule capable of hybridizing to a secondRNA molecule.

The term “homology” refers to the level of similarity between two ormore nucleic acid or amino acid sequences in terms of percent ofpositional identity (i.e., sequence similarity or identity). Homologyalso refers to the concept of similar functional properties amongdifferent nucleic acids or proteins.

The term “heterologous” refers to the relationship between two or morenucleic acid or protein sequences that are derived from differentsources. For example, a promoter is heterologous with respect to acoding sequence if such a combination is not normally found in nature.In addition, a particular sequence may be “heterologous” with respect toa cell or organism into which it is inserted (i.e., does not naturallyoccur in that particular cell or organism).

The term “hybridization” refers to the ability of a first strand ofnucleic acid to join with a second strand via hydrogen bond base pairingwhen the two nucleic acid strands have sufficient sequence identity.Hybridization occurs when the two nucleic acid molecules anneal to oneanother under appropriate conditions.

The phrase “operably linked” refers to the functional spatialarrangement of two or more nucleic acid regions or nucleic acidsequences. For example, a promoter region may be positioned relative toa nucleic acid sequence such that transcription of a nucleic acidsequence is directed by the promoter region. Thus, a promoter region is“operably linked” to the nucleic acid sequence.

The term or phrase “promoter” or “promoter region” refers to a nucleicacid sequence, usually found upstream (5′) to a coding sequence, that iscapable of directing transcription of a nucleic acid sequence into mRNA.The promoter or promoter region typically provides a recognition sitefor RNA polymerase and the other factors necessary for proper initiationof transcription. As contemplated herein, a promoter or promoter regionincludes variations of promoters derived by inserting or deletingregulatory regions, subjecting the promoter to random or site-directedmutagenesis, etc. The activity or strength of a promoter may be measuredin terms of the amounts of RNA it produces, or the amount of proteinaccumulation in a cell or tissue, relative to a promoter whosetranscriptional activity has been previously assessed.

The phrase “5′ UTR” refers to the untranslated region of DNA upstream,or 5′ of the coding region of a gene.

The phrase “3′ UTR” refers to the untranslated region of DNA downstream,or 3′ of the coding region of a gene.

The phrase “recombinant vector” refers to any agent such as a plasmid,cosmid, virus, autonomously replicating sequence, phage, or linearsingle-stranded, circular single-stranded, linear double-stranded, orcircular double-stranded DNA or RNA nucleotide sequence. The recombinantvector may be derived from any source and is capable of genomicintegration or autonomous replication.

The phrase “regulatory sequence” refers to a nucleotide sequence locatedupstream (5′), within, or downstream (3′) to a coding sequence.Transcription and expression of the coding sequence is typicallyimpacted by the presence or absence of the regulatory sequence.

The phrase “substantially homologous” refers to two sequences which areat least about 90% identical in sequence, as measured by the BestFitprogram described herein (Version 10; Genetics Computer Group, Inc.,University of Wisconsin Biotechnology Center, Madison, Wis.), usingdefault parameters.

The term “transformation” refers to the introduction of nucleic acidinto a recipient host. The term “host” refers to bacteria cells, fungi,animals or animal cells, plants or seeds, or any plant parts or tissuesincluding plant cells, protoplasts, calli, roots, tubers, seeds, stems,leaves, seedlings, embryos, and pollen.

As used herein, the phrase “transgenic plant” refers to a plant havingan introduced nucleic acid stably introduced into a genome of the plant,for example, the nuclear or plastid genomes.

As used herein, the phrase “substantially purified” refers to a moleculeseparated from substantially all other molecules normally associatedwith it in its native state. More preferably a substantially purifiedmolecule is the predominant species present in a preparation. Asubstantially purified molecule may be greater than about 60% free,preferably about 75% free, more preferably about 90% free, and mostpreferably about 95% free from the other molecules (exclusive ofsolvent) present in the natural mixture. The phrase “substantiallypurified” is not intended to encompass molecules present in their nativestate.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides promoters capable of transcribing aheterologous structural nucleic acid sequence in a seed, and methods ofmodifying, producing, and using the same. The present invention alsoprovides compositions, transformed host cells, and plants containingseed specific promoters, and methods for preparing and using the same.

Nucleic Acid Molecules

The present invention provides nucleic acid molecules comprising asequence selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4,9, 10, and 11, and complements thereof. SEQ ID NO: 5 represents areported USP promoter.

Nucleic acid hybridization is a technique well known to those of skillin the art of DNA manipulation. The hybridization property of a givenpair of nucleic acids is an indication of their similarity or identity.

Low stringency conditions may be used to select nucleic acid sequenceswith lower sequence identities to a target nucleic acid sequence. Onemay wish to employ conditions such as about 0.15 M to about 0.9 M sodiumchloride, at temperatures ranging from about 20° C. to about 55° C.

High stringency conditions may be used to select for nucleic acidsequences with higher degrees of identity to the disclosed nucleic acidsequences (Sambrook et al., 1989).

The high stringency conditions typically involve nucleic acidhybridization in about 2× to about 10×SSC (diluted from a 20×SSC stocksolution containing 3 M sodium chloride and 0.3 M sodium citrate, pH 7.0in distilled water), about 2.5× to about 5× Denhardt's solution (dilutedfrom a 50× stock solution containing 1% (w/v) bovine serum albumin, 1%(w/v) ficoll, and 1% (w/v) polyvinylpyrrolidone in distilled water),about 10 mg/mL to about 100 mg/mL fish sperm DNA, and about 0.02% (w/v)to about 0.1% (w/v) SDS, with an incubation at about 50° C. to about 70°C. for several hours to overnight. The high stringency conditions arepreferably provided by 6×SSC, 5× Denhardt's solution, 100 mg/mL fishsperm DNA, and 0.1% (w/v) SDS, with an incubation at 55° C. for severalhours.

The hybridization is generally followed by several wash steps. The washcompositions generally comprise 0.5× to about 10×SSC, and 0.01% (w/v) toabout 0.5% (w/v) SDS with a 15 minute incubation at about 20° C. toabout 70° C. Preferably, the nucleic acid segments remain hybridizedafter washing at least one time in 0.1×SSC at 65° C.

The nucleic acid molecules of the present invention preferablyhybridize, under high stringency conditions, with a nucleic acidmolecule having the sequence selected from the group consisting of SEQID NOS: 1, 2, 3, 4, 9, 10, and 11, and complements thereof. In apreferred embodiment, a nucleic acid molecule of the present inventionhybridizes, under high stringency conditions, with a nucleic acidmolecule comprising SEQ ID NO: 1. In a preferred embodiment, a nucleicacid molecule of the present invention hybridizes, under high stringencyconditions, with a nucleic acid molecule comprising SEQ ID NO: 2. In apreferred embodiment, a nucleic acid molecule of the present inventionhybridizes, under high stringency conditions, with a nucleic acidmolecule comprising SEQ ID NO: 3. In a preferred embodiment, a nucleicacid molecule of the present invention hybridizes, under high stringencyconditions, with a nucleic acid molecule comprising SEQ ID NO: 4. In apreferred embodiment, a nucleic acid molecule of the present inventionhybridizes, under high stringency conditions, with a nucleic acidmolecule comprising SEQ ID NO: 9. In a preferred embodiment, a nucleicacid molecule of the present invention hybridizes, under high stringencyconditions, with a nucleic acid molecule comprising SEQ ID NO: 10. In apreferred embodiment, a nucleic acid molecule of the present inventionhybridizes, under high stringency conditions, with a nucleic acidmolecule comprising SEQ ID NO: 11.

In a preferred embodiment, a nucleic acid molecule of the presentinvention comprises a nucleic acid sequence that has a sequence identityto SEQ ID NO: 1 of greater than about 85.5%, or greater than about 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99%.

In a preferred embodiment, a nucleic acid molecule of the presentinvention comprises a nucleic acid sequence that has a sequence identityto SEQ ID NO: 2 of greater than about 85.5%, or greater than about 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99%.

In a preferred embodiment, a nucleic acid molecule of the presentinvention comprises a nucleic acid sequence that has a sequence identityto SEQ ID NO: 3 of greater than about 97.1%, 98, 98.5, or greater thanabout 99%.

In a preferred embodiment, a nucleic acid molecule of the presentinvention comprises a nucleic acid sequence that has a sequence identityto SEQ ID NO: 4 of greater than about 96.4%, 97, 98, or about 99%.

The percent of sequence identity is preferably determined using thefollowing method. A sequence is converted to EditSeq DNA sequence filesusing EditSeq application program in the Dnastar software package(DNASTAR, Inc., Madison, Wis.). Converted files are imported into theMegalign application program of the Dnastar software package. Theimported sequences are aligned using the Clustal method at the defaultsetting with weighted residue weight table. This method is used todetermine the percent identity of the promoter sequences of the presentinvention to other sequences and to each other.

An alternative method for determining percent identity uses the “BestFit” or “Gap” program of the Sequence Analysis Software Package™(Version 10; Genetics Computer Group, Inc., University of WisconsinBiotechnology Center, Madison, Wis.). “Gap” utilizes the algorithm ofNeedleman and Wunsch (1970) to find the alignment of two sequences thatmaximizes the number of matches and minimizes the number of gaps.“BestFit” performs an optimal alignment of the best segment ofsimilarity between two sequences and inserts gaps to maximize the numberof matches using the local homology algorithm of Smith and Waterman(Smith and Waterman, 1981; Smith et al., 1983). The percent identity ismost preferably determined using the “Best Fit” program using defaultparameters.

The present invention also provides nucleic acid molecule fragments thatexhibit a percent identity to any of SEQ ID NOS: 1, 2, 3, 4, 9, 10, and11, and complements thereof that is greater than the percent identitythe fragments show to SEQ ID NO: 5. In a preferred embodiment, thepercent identity of a fragment to any of SEQ ID NOS: 1 through 4 is atleast about 1% greater than the percent identity of that fragment to SEQID NO: 5, and preferably is at least about 2, 3, 4, 5, 10, 15, 20, orabout 30% greater.

In an embodiment, the fragments are between about 50 and about 600consecutive nucleotides, about 50 and about 550 consecutive nucleotides,about 50 and about 500 consecutive nucleotides, about 50 and about 450consecutive nucleotides, about 50 and about 400 consecutive nucleotides,about 50 and about 350 consecutive nucleotides, about 50 and about 300consecutive nucleotides, about 50 and about 250 consecutive nucleotides,about 50 and about 200 consecutive nucleotides, about 50 and about 150consecutive nucleotides, about 50 and about 100, about 15 to about 100,about 15 to about 50, or about 15 to about 25 consecutive nucleotides ofa nucleic molecule of the present invention.

In another embodiment, the fragment comprises at least about 15, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, or about 650 consecutive nucleotides of a nucleic acidsequence of the present invention.

The present invention contemplates nucleic acid sequences encodingpolypeptides having the enzyme activity of the steroid pathway enzymessqualene epoxidase, sterol methyl transferase I, sterol C4 demethylase,obtusifoliol C14 α demethylase, sterol C5 desaturase, and sterol methyltransferase II.

Squalene epoxidase (also called squalene monooxygenase) catalyzes theconversion of squalene to squalene epoxide (2,3-oxidosqualene), aprecursor to the initial sterol molecule in phytosterol biosyntheticpathway, cycloartenol. This is the first reported step in the pathwaywhere oxygen is required for activity. The formation of squalene epoxideis also the last common reported step in sterol biosynthesis of animals,fungi, and plants. Recently, several homologues of Arabidopsis andBrassica squalene epoxidase genes were reported (Schafer, U. A., Reed,D. W., Hunter, D. G., Yao, K., Weninger, A. M., Tsang, E. W., Reaney, M.J., MacKenzie, S. L., and Covello, P. S. (1999), Plant Mol. Biol.,39(4):721–728). The same authors also have PCT application disclosingthe use of antisense technology with squalene epoxidase to elevatesqualene levels in plants (WO 97/34003).

Squalene Epoxidase, also known as squalene monooxygenase is enzymereference number 1.14.99.7, Enzyme Nomenclature, 1992, p. 146.

Several squalene epoxidase enzymes are known to the art. These includeArabidopsis squalene epoxidase protein sequence Accession No. AC004786Arabidopsis squalene epoxidase Accession No. N64916, and Arabidopsissqualene epoxidase Accession No. T44667. Japanese Patent Application No.07194381 A, discloses a DNA encoding a mammalian squalene epoxidase.

An additional aspect of the present invention is the recombinantconstructs and vectors comprising nucleic acid sequences encodingsqualene epoxidase, as well as a method of producing the novel squaleneepoxidase, comprising culturing a host cell transformed with the novelconstructs or vectors for a time and under conditions conductive to theproduction of the squalene epoxidase, and recovering the squaleneepoxidase produced thereby.

S-adenosyl L-methionine:sterol C24 methyl transferases (SMT1 and SMT2)catalyze the transfer of a methyl group from a cofactor,S-adenosyl-L-methionine, to the C24 center of the sterol side chain(Bach, T. J. and Benveniste, P. (1997), Prog. Lipid Res., 36:197–226).SMT in higher plant cells are responsible for their capability toproduce a mixture of 24-methyl and 24-ethyl sterols (Schaffer, A.,Bouvier-Navé, Benveniste, P., Schaller, H. (2000) Lipids, 35:263–269).Functional characterization of the SMT using a yeast erg6 expressionsystem demonstrated unambiguously that an SMT1 sequence encodes acycloartenol-C24-methyltransferase and a SMT2 sequence encodes aC24-methylene lophenol-C24-methyltransferase in a given plant species(Bouvier-Navé, P., Husselstein, T., and Benveniste, P. (1998), Eur. J.Biochem., 246:518–529). Several plant genes coding for SMT1 and SMT2have been reported and reviewed (Schaffer, A., Bouvier-Navé, Benveniste,P., Schaller, H. (2000) Lipids, 35:263–269). Transgenic plantsexpressing homologues of either SMT1 or SMT2 have been studied(Schaffer, A., Bouvier-Navé, Benveniste, P., Schaller, H. (2000) Lipids,35:263–269). The use of these genes to modify plant sterol compositionare also covered by two patent applications (WO 98/45457 and WO00/61771).

Sterol methyl transferase I enzymes known in the art are useful in thepresent invention. Exemplary sequences include the known Arabidopsissterol methyl transferase I protein sequence Accession No. U71400(disclosure SEQ ID NO: 19), the known tobacco sterol methyl transferaseI protein sequence Accession No. U81312 (disclosure SEQ ID NO: 20) andRicinus communis sterol C methyltransferase, Eur. J. Biochem.,246(2):518–529 (1997). (Complete cds, Accession No. g2246457).

S-Adenosyl L-Methionine Sterol C24 Methyltransferase—A nucleic acidsequence encoding an Arabidopsis thaliana S-adenosyl-L-methionine sterolC24 methyltransferase has been published by Husselstein et al., (1996)FEBS Letters 381:87–92. Δ²⁴ sterol methyltransferase is enzyme number2.1.1.41, Enzyme Nomenclature, 1992, p. 160.

Sterol C4 demethylase catalyses the first of several demethylationreactions, which results in the removal of the two methyl groups at C4.While in animals and fungi the removal of the two C4 methyl groupsoccurs consecutively, in plants it has been reported that there areother steps between the first and second C4 demethylations (Bach, T. J.and Benveniste, P. (1997), Prog. Lipid Res., 36:197–226). The C4demethylation is catalyzed by a complex of microsomal enzymes consistingof a monooxygenase, an NAD⁺-dependent sterol 4-decarboxylase and anNADPH-dependent 3-ketosteroid reductase.

Sterol C14 demethylase catalyzes demethylation at C14 which removes themethyl group at C14 and creates a double bond at that position. In bothfungi and animals, this is the first step in the sterol synthesispathway. However, in higher plants, the 14α-methyl is removed after oneC4 methyl has disappeared. Thus, while lanosterol is the substrate forC4 demethylase in animal and fungal cells, the plants enzyme usesobtusifoliol as substrate. Sterol C14 demethylation is mediated by acytochrome P-450 complex. The mechanism of 14-methyl removal involvestwo oxidation steps leading to an alcohol, then an aldehyde at C29 and afurther oxidative step involving a deformylation leading to formic acidand the sterol product with a typical 8, 14-diene (Aoyama, Y., Yoshida,Y., Sonoda, Y., and Sato, Y. (1989) J. Biol. Chem., 264:18502–18505).Obtusifoliol C14 α-demethylase from Sorghum bicolor (L) Moench has beencloned using a gene-specific probe generated using PCR primers designedfrom an internal 14 amino acid sequence and was functionally expressedin E. coli (Bak, S., Kahn, R. A., Olsen, C. E., and Halkier, B. A.(1997) The Plant Journal, 11(2):191–201). Also, Saccharomyces cerevisiaeCYP51A1 encoding lanosterol 14-demethylase was functionally expressed intobacco (Grausem, B., Chaubet, N., Gigot, C., Loper, J. C., andBenveniste, P. (1995) The Plant Journal, 7(5):761–770).

Sterol C14 demethylase enzymes and sequences are known in the art. Forexample Sorghum bicolor obtusifoliol C14 α-demethylase CYP51 mRNA,described in Plant J., 11(2):191–201 (1997) (complete cds Acession No.U74319).

An additional aspect of the present invention is the recombinantconstructs and vectors comprising nucleic acid sequences encoding thenovel obtusifoliol C14 α-demethylase, as well as a method of producingthe novel obtusifoliol C14 α-demethylase, comprising culturing a hostcell transformed with the novel constructs or vectors for a time andunder conditions conductive to the production of the obtusifoliol C14α-demethylase, and recovering the obtusifoliol C14 α-demethylaseproduced thereby.

Sterol C5 desaturase catalyzes the insertion of the Δ⁵-double bond thatnormally occurs at the Δ⁷-sterol level, thereby forming a Δ^(5,7)-sterol(Parks et al., Lipids, 30:227–230 (1995)). The reaction has beenreported to involve the stereospecific removal of the 5α and 6α hydrogenatoms, biosynthetically derived from the 4 pro-R and 5 pro-S hydrogensof the (+) and (−) R-mevalonic acid, respectively (Goodwin, T. W. (1979)Annu. Rev. Plant Physiol., 30:369–404). The reaction is obligatorilyaerobic and requires NADPH or NADH. The desaturase has been reported tobe a multienzyme complex present in microsomes. It consists of thedesaturase itself, cytochrome b₅ and a pyridine nucleotide-dependentflavoprotein. The Δ⁵-desaturase is reported to be a mono-oxygenase thatutilizes electrons derived from a reduced pyridine nucleotide viacytochrome_(b) (Taton, M., and Rahier, A. (1996) Arch. Biochem.Biophys., 325:279–288). An Arabidopsis thaliana cDNA encoding asterol-C5 desaturase was cloned by functional complementation of a yeastmutant, erg3 defective in ERG3, the gene encoding the sterol C5desaturase required for ergosterol biosynthesis (Gachotte D.,Husselstein, T., Bard, M., Lacroute F., and Benveniste, P. (1996) ThePlant Journal, 9(3):391–398). Known sterol C5 desaturase enzymes areuseful in the present invention, including Arabidopsis sterol C5desaturase protein sequence Accession No. X90454, disclosure SEQ ID NO:22, and the Arabidopsis thaliana mRNA for sterol C5 desaturase describedin The Plant J. 9(3):391-398 (1996) (complete cds Accession No.g1061037).

The NCBI (National Center for Biotechnology Information) database shows37 sequences for sterol desaturase that are useful in the presentinvention. The following are exemplary of such sequences. From yeast: C5sterol desaturase NP_(—)013157 (Saccharomyces cerevisiae); hypotheticalC5 sterol desaturase-fission T40027 (Schizosaccharomyces pombe); C5sterol desaturase-fission T37759 (Schizosaccharomyces pombe); C5 steroldesaturase JQ1146 (Saccharomyces cerevisiae); C5 sterol desaturaseBAA21457 (schizosaccharomyces pombe); C5 sterol desaturase CAA22610(Schizosaccharomyces pombe); putative C5 sterol desaturase CAA16898(Schizosaccharomyces pombe); probable C5 sterol desaturase O13666(erg3_schpo); C5 sterol desaturase P50860 (Erg3_canga); C5 steroldesaturase P32353 (erg3_yeast); C5,6 desaturase AAC99343 (Candidaalbicans); C5 sterol desaturase BAA20292 (Saccharomyces cerevisiae); C5sterol desaturase AAB39844 (Saccharomyces cerevisiae); C5 steroldesaturase AAB29844 (Saccharomyces cerevisiae); C5 sterol desaturaseCAA64303 (Saccharomyces cerevisiae); C5 sterol desaturase AAA34595(Saccharomyces cerevisiae); C5 sterol desaturase AAA34594 (Saccharomycescerevisiae). From plants: C5 sterol desaturase S71251 (Arabidopsisthaliana); putative sterol C5 desaturase AAF32466 (Arabidopsisthaliana); sterol C5 desaturase AAF32465 (Arabidopsis thaliana);putatuve sterol desaturase AAF22921 (Arabidopsis thaliana); Δ⁷-sterol C5desaturase (Arabidopsis thaliana); sterol C5,6 desaturase homologAAD20458 (Nicotiana tabacum); sterol C5 desaturase AAD12944 (Arabidopsisthaliana); sterol C5,6 desaturase AAD04034 (Nicotiana tabacum); sterolC5 desaturase CAA62079 (Arabidopsis thaliana). From mammals: sterol C5desaturase (Mus musculus) BAA33730; sterol C5 desaturase BAA33729 (Homosapiens); lathosterol oxidase CAB65928 (Leishmania major); lathosteroloxidase (lathosterol C5 desaturase) 088822 (Mus musculus); lathosterolC5 desaturase 075845 (Homo sapiens); Δ⁷-sterol C5 desaturase AAF00544(Homo sapiens). Others: fungal sterol C5 desaturase homolog BAA18970(Homo sapiens).

For DNA sequences encoding a sterol-C5 desaturase useful in the presentinvention, the NCBI_nucleotide search for “sterol desaturase” came upwith 110 sequences. The following are exemplary of such sequences.NC_(—)001139 (Saccharomyces cerevisiae); NC_(—)001 145 (Saccharomycescerevisiae); NC_(—)001144 (Saccharomyces cerevisiae); AW700015(Physcomitrella patens); AB004539 (Schizosaccharomyces pombe); andAW596303 (Glycine max); AC012188 (Arabidopsis thaliana).

The combination of introduction of an HMG-CoA reductase gene along witha sterol methyl transferase II gene into a cell serves to reduce steroidpathway intermediate compound accumulation in addition to reducing theaccumulation of 24-methyl sterols such as campesterol.

Known sterol methyl transferase II enzymes are useful in the presentinvention, including Arabidopsis sterol methyl transferase II proteinsequence (complete mRNA cds from FEBS Lett. 381(12):87–92 (1996)Accession No. X89867), disclosure SEQ ID NO: 21.

Recombinant constructs encoding any of the forgoing enzymes affectingthe steroid biosynthetic pathway can be incorporated into recombinantvectors comprising the recombinant constructs comprising the isolatedDNA molecules. Such vectors can be bacterial or plant expressionvectors.

In a preferred embodiment, any of the plants or organisms of the presentinvention are transformed with a nucleic acid of the present inventionand a gene encoding a member selected from the group consisting ofsqualene epoxidase, sterol methyl transferase I, sterol C4 demethylase,obtusifoliol C14 α-demethylase, sterol C5 desaturase, and sterol methyltransferase II. In a preferred embodiment, a plant or organism of thepresent invention is transformed with one or more of SEQ ID NOS: 1–5 anda gene encoding a member selected from the group consisting of squaleneepoxidase, sterol methyl transferase I, sterol C4 demethylase,obtusifoliol C14 α-demethylase, sterol C5 desaturase, and sterol methyltransferase II. In a further preferred embodiment, a plant or organismof the present invention is transformed with one or more of SEQ ID NOS:1–5, a gene encoding a member selected from the group consisting ofsqualene epoxidase, sterol methyl transferase I, sterol C4 demethylase,obtusifoliol C14 α-demethylase, sterol C5 desaturase, and sterol methyltransferase II, and one or more genes encoding a tocopherol pathwayenzyme as disclosed elsewhere herein. In a further preferred embodiment,a plant or organism of the present invention is transformed with one ormore of SEQ ID NOS: 1–5, two genes encoding a member selected from thegroup consisting of squalene epoxidase, sterol methyl transferase I,sterol C4 demethylase, obtusifoliol C14 α-demethylase, sterol C5desaturase, and sterol methyl transferase II, and two genes encoding atocopherol pathway enzyme as disclosed elsewhere herein. Any of theabove combinations of tocopherol and sterol biosynthesis genes can beintroduced into a plant on one or more constructs or vectors, as isknown in the art and described herein.

Promoters

In one embodiment any of the disclosed nucleic acid molecules may bepromoters. In a preferred embodiment, the promoter is tissue or organspecific, and preferably seed specific. In a particularly preferredembodiment the promoter preferentially expresses associated structuralgenes in the endosperm or embryo. In a preferred embodiment, thepromoter is a USP promoter. In a particularly preferred embodiment, thepromoter is a Vicia faba USP promoter.

In one aspect, a promoter is considered tissue or organ specific if thelevel of an mRNA in that tissue or organ is expressed at a level that isat least 10 fold higher, preferably at least 100 fold higher or at least1,000 fold higher than another tissue or organ. The level of mRNA can bemeasured either at a single time point or at multiple time points and assuch the fold increase can be average fold increase or an extrapolatedvalue derived from experimentally measured values. As it is a comparisonof levels, any method that measures mRNA levels can be used. In apreferred aspect, the tissue or organs compared are a seed or seedtissue with a leaf or leaf tissue. In another preferred aspect, multipletissues or organs are compared. A preferred multiple comparison is aseed or seed tissue compared with 2, 3, 4, or more tissues or organsselected from the group consisting of floral tissue, floral apex,pollen, leaf, embryo, shoot, leaf primordia, shoot apex, root, root tip,vascular tissue and cotyledon. As used herein, examples of plant organsare seed, leaf, root, etc. and example of tissues are leaf primordia,shoot apex, vascular tissue, etc.

The activity or strength of a promoter may be measured in terms of theamount of mRNA or protein accumulation it specifically produces,relative to the total amount of mRNA or protein. The promoter preferablyexpresses an operably linked nucleic acid sequence at a level greaterthan about 2.5%; more preferably greater than about 5, 6, 7, 8, or about9%; even more preferably greater than about 10, 11, 12, 13, 14, 15, 16,17, 18, or about 19%, and most preferably greater than about 20% of thetotal mRNA.

Alternatively, the activity or strength of a promoter may be expressedrelative to a well-characterized promoter (for which transcriptionalactivity was previously assessed). For example, a promoter of interestmay be operably linked to a reporter sequence (e.g., GUS) and introducedinto a specific cell type. A known promoter may be similarly preparedand introduced into the same cellular context. Transcriptional activityof the promoter of interest is then determined by comparing the amountof reporter expression, relative to the known promoter. The cellularcontext is preferably soybean.

Structural Nucleic Acid Sequences

The promoters of the present invention may be operably linked to astructural nucleic acid sequence that is heterologous with respect tothe promoter. The structural nucleic acid sequence may generally be anynucleic acid sequence for which an increased level of transcription isdesired. The structural nucleic acid sequence preferably encodes apolypeptide that is suitable for incorporation into the diet of a humanor an animal or provides some other agriculturally important feature.

Suitable structural nucleic acid sequences include, without limitation,those encoding seed storage proteins, fatty acid pathway enzymes,tocopherol biosynthetic enzymes, amino acid biosynthetic enzymes,steroid pathway enzymes, and starch branching enzymes.

Preferred seed storage proteins include zeins (U.S. Pat. Nos. 4,886,878;4,885,357; 5,215,912; 5,589,616; 5,508,468; 5,939,599; 5,633,436; and5,990,384; Patent Applications: WO 90/01869, WO 91/13993, WO 92/14822,WO 93/08682, WO 94/20628, WO 97/28247, WO 98/26064, and WO 99/40209), 7Sproteins (U.S. Pat. Nos. 5,003,045 and 5,576,203), brazil nut protein(U.S. Pat. No. 5,850,024), phenylalanine free proteins (PatentApplication: WO 96/17064), albumin (Patent Application: WO 97/35023),β-conglycinin (Patent Application: WO 00/19839), 11S (U.S. Pat. No.6,107,051), (α-hordothionin (U.S. Pat. Nos. 5,885,802 and 5885801),arcelin seed storage proteins (U.S. Pat. No. 5,270,200), lectins (U.S.Pat. No. 6,110,891), and glutenin (U.S. Pat. Nos. 5,990,389 and5,914,450).

Preferred fatty acid pathway enzymes include thioesterases (U.S. Pat.Nos. 5,512,482; 5,530,186; 5,945,585; 5,639,790; 5,807,893; 5,955,650;5,955,329; 5,759,829; 5,147,792; 5,304,481; 5,298,421; 5,344,771; and5,760,206), and desaturases (U.S. Pat. Nos. 5,689,050; 5,663,068;5,614,393; 5,856,157; 6,117,677; 6,043,411; 6,194,167; 5,705,391;5,663,068; 5,552,306; 6,075,183; 6,051,754; 5,689,050; 5,789,220;5,057,419; 5,654,402; 5,659,645; 6,100,091; 5,760,206; 6,172,106;5,952,544; 5,866,789; 5,443,974; and 5,093,249). Preferred tocopherolbiosynthetic enzymes include tyrA, slr1736, ATPT2, dxs, dxr, GGPPS,HPPD, GMT, MT1, tMT2, AANT1, slr1737, and an antisense construct forhomogentisic acid dioxygenase (Krid1 et al., Seed Sci. Res., 1:209:219(1991); Keegstra, Cell, 56(2):247–53 (1989); Nawrath, et al., Proc.Natl. Acad. Sci. (U.S.A.), 91:12760–12764 (1994); Xia et al., J. Gen.Microbiol., 138:1309–1316 (1992); Cyanobase www.kazusa.or.jp/cyanobase;Lois et al., Proc. Natl. Acad. Sci. (U.S.A.), 95(5):2105–2110 (1998);Takahashi et al., Proc. Natl. Acad. Sci. (U.S.A.), 95(17), 9879–9884(1998); Norris et al., Plant Physiol., 117:1317–1323 (1998); Bartley andScolnik, Plant Physiol., 104:1469–1470 (1994); Smith et al., Plant J.,11:83–92 (1997); WO 00/32757; WO 00/10380; Saint Guily, et al., PlantPhysiol., 100(2):1069–1071 (1992); Sato et al, J. DNA Res., 7(1):31–63(2000)).

Various genes and their encoded proteins that are involved in tocopherolbiosynthesis are listed in Table 1 below.

TABLE 1 Genes and Encoded Proteins Involved in Tocopherol BiosynthesisGene ID Enzyme name tyrA Prephanate dehydrogenase slr1736 Phytylprenyltransferase from Synechocystis ATPT2 Phytylprenyl transferase fromArabidopsis thaliana DXS 1-Deoxyxylulose-5-phosphate synthase DXR1-Deoxyxylulose-5-phosphate reductoisomerase GGPPS Geranylgeranylpyrophosphate synthase HPPD p-Hydroxyphenylpyruvate dioxygenase AANT1Adenylate transporter slr1737 Tocopherol cyclase IDI Isopentenyldiphosphate isomerase GGH Geranylgeranyl reductase GMT Gamma MethylTransferase

The “Gene IDs” given in Table 1 above identify the gene associated withthe listed 10 enzyme. Any of the Gene IDs listed in Table 1 appearingherein in the present disclosure refer to the gene encoding the enzymewith which the Gene ID is associated in Table 1.

Preferred amino acid biosynthetic enzymes include anthranilate synthase(U.S. Pat. No. 5,965,727; Patent Applications: WO 97/26366, WO 99/11800,and WO 99/49058), tryptophan decarboxylase (Patent Application: WO99/06581), threonine decarboxylase (U.S. Pat. Nos. 15 5,534,421 and5,942,660, Patent Application: WO 95/19442), threonine deaminase (PatentApplications: WO 99/02656, and WO 98/55601), and aspartate kinase (U.S.Pat. Nos. 5,367,110; 5,858,749; and 6,040,160).

Preferred starch branching enzymes include those set forth in U.S. Pat.Nos. 6,232,122 and 6,147, 279; and Patent Application WO 97/22703.

Alternatively, a promoter and structural nucleic acid sequence may bedesigned to down-regulate a specific nucleic acid sequence. This istypically accomplished by linking the promoter to a structural nucleicacid sequence that is oriented in the antisense direction. One ofordinary skill in the art is familiar with such antisense technology.Any nucleic acid sequence may be negatively regulated in this manner.

Targets of such regulation may include polypeptides that have a lowcontent of essential amino acids, yet are expressed at a relatively highlevel in a particular tissue. For example, β-conglycinin and glycininare expressed abundantly in seeds, but are nutritionally deficient withrespect to essential amino acids. This antisense approach may also beused to effectively remove other undesirable proteins, such asantifeedants (e.g., lectins), albumin, and allergens, from plant-derivedfeed or to down-regulate catabolic enzymes involved in degradation ofdesired compounds such as essential amino acids.

Modified Structural Nucleic Acid Sequences

The promoters of the present invention may also be operably linked to amodified structural nucleic acid sequence that is heterologous withrespect to the promoter. The structural nucleic acid sequence may bemodified to provide various desirable features. For example, astructural nucleic acid sequence may be modified to increase the contentof essential amino acids, enhance translation of the amino acidsequence, alter post-translational modifications (e.g., phosphorylationsites), transport a translated product to a compartment inside oroutside of the cell, improve protein stability, insert or delete cellsignaling motifs, etc.

In a preferred embodiment, the structural nucleic acid sequence isenhanced to encode a polypeptide having an increased content of at leastone, and more preferably 2, 3, or 4 of the essential amino acidsselected from the group consisting of histidine, lysine, methionine, andphenylalanine. Non-essential amino acids may also be added, as needed,for structural and nutritive enhancement of the polypeptide. Structuralnucleic acid sequences particularly suited to such enhancements includethose encoding native polypeptides that are expressed at relatively highlevels, have a particularly low content of essential amino acids, orboth. An example of such are the seed storage proteins, such as glycininand β-conglycinin. Other suitable targets include arcelin, phaseolin,lectin, zeins, and albumin.

Codon Usage in Structural Nucleic Acid Sequences

Due to the degeneracy of the genetic code, different nucleotide codonsmay be used to code for a particular amino acid. A host cell oftendisplays a preferred pattern of codon usage. Structural nucleic acidsequences are preferably constructed to utilize the codon usage patternof the particular host cell. This generally enhances the expression ofthe structural nucleic acid sequence in a transformed host cell. Any ofthe above described nucleic acid and amino acid sequences may bemodified to reflect the preferred codon usage of a host cell or organismin which they are contained. Modification of a structural nucleic acidsequence for optimal codon usage in plants is described in U.S. Pat. No.5,689,052.

Other Modifications of Structural Nucleic Acid Sequences

Additional variations in the structural nucleic acid sequences describedabove may encode proteins having equivalent or superior characteristicswhen compared to the proteins from which they are engineered. Mutationsmay include deletions, insertions, truncations, substitutions, fusions,shuffling of motif sequences, and the like.

Mutations to a structural nucleic acid sequence may be introduced ineither a specific or random manner, both of which are well known tothose of skill in the art of molecular biology. A myriad ofsite-directed mutagenesis techniques exist, typically usingoligonucleotides to introduce mutations at specific locations in astructural nucleic acid sequence. Examples include single strand rescue(Kunkel et al., 1985), unique site elimination (Deng and Nickloff,1992), nick protection (Vandeyar et al., 1988), and PCR (Costa et al.,1996). Random or non-specific mutations may be generated by chemicalagents (for a general review, see Singer and Kusmierek, 1982) such asnitrosoguanidine (Cerda-Olmedo et al., 1968; Guerola et al., 1971), and2-aminopurine (Rogan and Bessman, 1970); or by biological methods suchas passage through mutator strains (Greener et al., 1997). Additionalmethods of making the alterations described above are described byAusubel et al. (1995); Bauer et al. (1985); Craik (1985); Frits Ecksteinet al. (1982); Sambrook et al. (1989); Smith et al. (1981); and Osuna etal. (1994).

The modifications may result in either conservative or non-conservativechanges in the amino acid sequence. Conservative changes are changeswhich do not alter the final amino acid sequence of the protein. In apreferred embodiment, the protein has between 5 and 500 conservativechanges, more preferably between 10 and 300 conservative changes, evenmore preferably between 25 and 150 conservative changes, and mostpreferably between 5 and 25 conservative changes or between 1 and 5conservative changes.

Non-conservative changes include additions, deletions, and substitutionswhich result in an altered amino acid sequence. In a preferredembodiment, the protein has between about 5 and about 500non-conservative amino acid changes, more preferably between about 10and about 300 non-conservative amino acid changes, even more preferablybetween about 25 and about 150 non-conservative amino acid changes, andmost preferably between about 5 and about 25 non-conservative amino acidchanges or between about 1 and about 5 non-5 conservative changes.

Modifications may be made to the protein sequences described herein andthe nucleic acid sequences that encode them that maintain the desiredproperties of the molecule. The following is a discussion based uponchanging the amino acid sequence of a protein to create an equivalent,or possibly an improved, second-generation molecule. The amino acidchanges may be achieved by changing the codons of the structural nucleicacid sequence, according to the codons given in Table 2.

TABLE 2 Codon degeneracy of amino acids One Three Amino acid letterLetter Codons Alanine A Ala GCA GCC GCG GCT Cysteine C Cys TGC TGTAspartic acid D Asp GAC GAT Glutamic acid E Glu GAA GAG Phenylalanine FPhe TTC TTT Glycine G Gly GGA GGC GGG GGT Histidine H His CAC CATIsoleucine I Ile ATA ATC ATT Lysine K Lys AAA AAG Leucine L Leu TTA TTGCTA CTC CTG CTT Methionine M Met ATG Asparagine N Asn AAC AAT Proline PPro CCA CCC CCG CCT Glutamine Q Gln CAA CAG Arginine R Arg AGA AGG CGACGC CGG CGT Serine S Ser AGC AGT TCA TCC TCG TCT Threonine T Thr ACA ACCACG ACT Valine V Val GTA GTC GTG GTT Tryptophan W Trp TGG Tyrosine Y TyrTAC TAT

Certain amino acids may be substituted for other amino acids in aprotein sequence without appreciable loss of the desired activity. It isthus contemplated that various changes may be made in peptide sequencesor protein sequences, or their corresponding nucleic acid sequenceswithout appreciable loss of the biological activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics. These are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−histidine (−3.2);glutamate/glutamine/aspartate/asparagine (−3.5); lysine (−3.9); andarginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biologically functional protein. In making such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those within ±1 are more preferred, and those within ±0.5 aremost preferred.

It is also understood in the art that the substitution of like aminoacids may be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein. The followinghydrophilicity values have been assigned to amino acids: arginine/lysine(+3.0); aspartate/glutamate (+3.0±1); serine (+0.3);asparagine/glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine/histidine (−0.5); cysteine (−1.0); methionine (−1.3);valine (−1.5); leucine/isoleucine (−1.8); tyrosine (−2.3); phenylalanine(−2.5); and tryptophan (−3.4).

It is understood that an amino acid may be substituted by another aminoacid having a similar hydrophilicity score and still result in a proteinwith similar biological activity, i.e., still obtain a biologicallyfunctional protein. In making such changes, the substitution of aminoacids whose hydropathic indices are within ±2 is preferred, those within±1 are more preferred, and those within ±0.5 are most preferred.

As outlined above, amino acid substitutions are therefore based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions which take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine, andisoleucine. Changes which are not expected to be advantageous may alsobe used if these resulted proteins have improved rumen resistance,increased resistance to proteolytic degradation, or both improved rumenresistance and increased resistance to proteolytic degradation, relativeto the unmodified polypeptide from which they are engineered.Alternatively, changes could be made to improve kinetics of metabolicenzymes.

In a preferred aspect, the protein modified is selected from seedstorage proteins, fatty acid pathway enzymes, tocopherol biosyntheticenzymes, amino acid biosynthetic enzymes and starch branching enzymes.

Recombinant Vectors

Any of the promoters and structural nucleic acid sequences describedabove may be provided in a recombinant vector. A recombinant vectortypically comprises, in a 5′ to 3′ orientation: a promoter to direct thetranscription of a structural nucleic acid sequence and a structuralnucleic acid sequence. Suitable promoters and structural nucleic acidsequences include those described herein. The recombinant vector mayfurther comprise a 3′ transcriptional terminator, a 3′ polyadenylationsignal, other untranslated nucleic acid sequences, transit and targetingnucleic acid sequences, selectable markers, enhancers, and operators, asdesired.

Means for preparing recombinant vectors are well known in the art.Methods for making recombinant vectors particularly suited to planttransformation are described in U.S. Pat. Nos. 4,971,908; 4,940,835;4,769,061; and 4,757,011. These types of vectors have also been reviewed(Rodriguez et al., 1988; Glick et al., 1993).

Typical vectors useful for expression of nucleic acids in higher plantsare well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens (Rogers et al.,1987). Other recombinant vectors useful for plant transformation,including the pCaMVCN transfer control vector, have also been described(Fromm et al., 1985).

In one embodiment, multiple USP promoters are operably linked in asingle construct to any combination of structural genes. In a preferredembodiment, any combination of 1, 2, 3, 4, 5, or 6 or more of nucleicacid molecules comprising SEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11 can beoperatively linked in a single construct to any combination ofstructural genes. In another aspect of the preferred embodiment, thenucleic acid molecules may be modified. Such modifications can include,for example, removal or addition of one or more structural or functionalelements.

Additional Promoters in the Recombinant Vector

One or more additional promoters may also be provided in the recombinantvector. These promoters may be operably linked, for example, withoutlimitation, to any of the structural nucleic acid sequences describedabove. Alternatively, the promoters may be operably linked to othernucleic acid sequences, such as those encoding transit peptides,selectable marker proteins, or antisense sequences.

These additional promoters may be selected on the basis of the cell typeinto which the vector will be inserted. Also, promoters which functionin bacteria, yeast, and plants are all well taught in the art. Theadditional promoters may also be selected on the basis of theirregulatory features. Examples of such features include enhancement oftranscriptional activity, inducibility, tissue specificity, anddevelopmental stage-specificity. In plants, promoters that areinducible, of viral or synthetic origin, constitutively active,temporally regulated, and spatially regulated have been described(Poszkowski et al., 1989; Odell et al., 1985; Chau et al., 1989).

Often-used constitutive promoters include the CaMV 35S promoter (Odellet al., 1985), the enhanced CaMV 35S promoter, the Figwort Mosaic Virus(FMV) promoter (Richins et al., 1987), the mannopine synthase (mas)promoter, the nopaline synthase (nos) promoter, and the octopinesynthase (ocs) promoter.

Useful inducible promoters include promoters induced by salicylic acidor polyacrylic acids (PR-1; Williams et al, 1992), induced byapplication of safeners (substituted benzenesulfonamide herbicides;Hershey and Stoner, 1991), heat-shock promoters (Ou-Lee et al., 1986;Ainley et al., 1990), a nitrate-inducible promoter derived from thespinach nitrite reductase structural nucleic acid sequence (Back et al.,1991), hormone-inducible promoters (Yamaguchi-Shinozaki et al., 1990;Kares et al., 1990), and light-inducible promoters associated with thesmall subunit of RuBP carboxylase and LHCP families (Kuhlemeier et al.,1989; Feinbaum et al., 1991; Weisshaar et al., 1991; Lam and Chua, 1990;Castresana et al., 1988; Schulze-Lefert et al., 1989).

Examples of useful tissue or organ specific promoters includeβ-conglycinin, (Doyle et al., 1986; Slighton and Beachy, 1987), andother seed specific promoters (Knutzon et al., 1992; Bustos et al.,1991; Lam and Chua, 1991). Plant functional promoters useful forpreferential expression in seed include those from plant storageproteins and from proteins involved in fatty acid biosynthesis inoilseeds. Examples of such promoters include the 5′ regulatory regionsfrom such structural nucleic acid sequences as napin (Kridl et al.,1991), phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACPdesaturase, and oleosin. Seed-specific regulation is further discussedin EP 0

Another exemplary seed specific promoter is a lectin promoter. Thelectin protein in soybean seeds is encoded by a single structuralnucleic acid sequence (Le1) that is only expressed during seeddevelopment. A lectin structural nucleic acid sequence and seed-specificpromoter have been characterized and used to direct seed specificexpression in transgenic tobacco plants (Vodkin et al., 1983; Lindstromet al., 1990).

Particularly preferred additional promoters in the recombinant vectorinclude the nopaline synthase (nos), mannopine synthase (mas), andoctopine synthase (ocs) promoters, which are carried on tumor-inducingplasmids of Agrobacterium tumefaciens; the cauliflower mosaic virus(CaMV) 19S and 35S promoters; the enhanced CaMV 35S promoter; theFigwort Mosaic Virus (FMV) 35S promoter; the light-inducible promoterfrom the small subunit of ribulose-1,5-bisphosphate carboxylase(ssRUBISCO); the EIF-4A promoter from tobacco (Mandel et al, 1995); cornsucrose synthetase 1 (Yang and Russell, 1990); corn alcoholdehydrogenase 1 (Vogel et al., 1989); corn light harvesting complex(Simpson, 1986); corn heat shock protein (Odell et al., 1985); thechitinase promoter from Arabidopsis (Samac et al., 1991); the LTP (LipidTransfer Protein) promoters from broccoli (Pyee et al., 1995); petuniachalcone isomerase (Van Tunen et al., 1988); bean glycine rich protein 1(Keller et al., 1989); potato patatin (Wenzler et al., 1989); theubiquitin promoter from maize (Christensen et al., 1992); and the actinpromoter from rice (McElroy et al., 1990).

An additional promoter is preferably seed selective, tissue selective,constitutive, or inducible. The promoter is most preferably the nopalinesynthase (nos), octopine synthase (ocs), mannopine synthase (mas),cauliflower mosaic virus 19S and 35S (CaMV19S, CaMV35S), enhanced CaMV(eCaMV), ribulose 1,5-bisphosphate carboxylase (ssRUBISCO), figwortmosaic virus (FMV), CaMV derived AS4, tobacco RB7, wheat POX1, tobaccoEIF-4, lectin protein (Le1), or rice RC2 promoter.

Recombinant Vectors Having Additional Structural Nucleic Acid Sequences

The recombinant vector may also contain one or more additionalstructural nucleic acid sequences. These additional structural nucleicacid sequences may generally be any sequences suitable for use in arecombinant vector. Such structural nucleic acid sequences include,without limitation, any of the structural nucleic acid sequences, andmodified forms thereof, described above. The additional structuralnucleic acid sequences may also be operably linked to any of the abovedescribed promoters. The one or more structural nucleic acid sequencesmay each be operably linked to separate promoters. Alternatively, thestructural nucleic acid sequences may be operably linked to a singlepromoter (i.e., a single operon).

The additional structural nucleic acid sequences include, withoutlimitation, those encoding seed storage proteins, fatty acid pathwayenzymes, tocopherol biosynthetic enzymes, amino acid biosyntheticenzymes, and starch branching enzymes.

Preferred seed storage proteins include zeins (U.S. Pat. Nos. 4,886,878;4,885,357; 5,215,912; 5,589,616; 5,508,468; 5,939,599; 5,633,436; and5,990,384; Patent Applications: WO 90/01869, WO 91/13993, WO 92/14822,WO 93/08682, WO 94/20628, WO 97/28247, WO 98/26064, and WO 99/40209), 7Sproteins (U.S. Pat. Nos. 5,003,045 and 5,576,203), brazil nut protein(U.S. Pat. No. 5,850,024), phenylalanine free proteins (PatentApplication: WO 96/17064), albumin (Patent Application: WO 97/35023),β-conglycinin (Patent Application: WO 00/19839), 11S (U.S. Pat. No.6,107,051), α-hordothionin (U.S. Pat. Nos. 5,885,802 and 5,88,5801)arcelin seed storage proteins (U.S. Pat. No. 5,270,200) lectins (U.S.Pat. No. 6,110,891) and glutenin (U.S. Pat. Nos. 5,990,389 and5,914,450).

Preferred fatty acid pathway enzymes include thioesterases (U.S. Pat.Nos. 5,512,482; 5,530,186; 5,945,585; 5,639,790; 5,807,893; 5,955,650;5,955,329; 5,759,829; 5,147,792; 5,304,481; 5,298,421; 5,344,771; and5,760,206), and desaturases (U.S. Pat. Nos. 5,689,050; 5,663,068;5,614,393; 5,856,157; 6,117,677; 6,043,411; 6,194,167; 5,705,391;5,663,068; 5,552,306; 6,075,183; 6,051,754; 5,689,050; 5,789,220;5,057,419; 5,654,402; 5,659,645; 6,100,091; 5,760,206; 6,172,106;5,952,544; 5,866,789; 5,443,974; and 5,093,249).

Preferred tocopherol biosynthetic enzymes include tyrA, slr1736, ATPT2,dxs, dxr, GGPPS, HPPD, GMT, MT1, tMT2, AANT1, slr1737, and an antisenseconstruct for homogentisic acid dioxygenase (Krid1et al., Seed Sci.Res., 1:209:219 (1991); Keegstra, Cell, 56(2):247–53 (1989); Nawrath, etal., Proc. Natl. Acad. Sci. (U.S.A.), 91:12760–12764 (1994); Xia et al.,J. Gen. Microbiol., 138:1309–1316 (1992); Cyanobasewww.kazusa.or.jp/cyanobase; Lois et al., Proc. Natl. Acad. Sci.(U.S.A.), 95(5):2105–2110 (1998); Takahashi et al. Proc. Natl. Acad.Sci. (U.S.A.), 95(17):9879–9884 (1998); Norris et al., Plant Physiol.,117:1317–1323 (1998); Bartley and Scolnik, Plant Physiol., 104:1469–1470(1994); Smith et al., Plant J., 11:83–92 (1997); WO 00/32757; WO00/10380; Saint Guily, et al., Plant Physiol., 100(2):1069–1071 (1992);Sato et al., J. DNA Res., 7(1):31–63 (2000)).

Preferred amino acid biosynthetic enzymes include anthranilate synthase(U.S. Pat. No. 5,965,727, Patent Applications: WO 97/26366, WO 99/11800,and WO 99/49058), tryptophan decarboxylase (Patent Application: WO99/06581), threonine decarboxylase (U.S. Pat. Nos. 5,534,421 and5,942,660; Patent Application: WO 95/19442), threonine deaminase (PatentApplications WO 99/02656 and WO 98/55601), and aspartate kinase (U.S.Pat. Nos. 5,367,110; 5,858,749; and 6,040,160).

Preferred starch branching enzymes include those set forth in U.S. Pat.Nos. 6,232,122 and 6,147,279; and Patent Application WO 97/22703.

Alternatively, the second structural nucleic acid sequence may bedesigned to down-regulate a specific nucleic acid sequence. This istypically accomplished by operably linking the second structural aminoacid, in an antisense orientation, with a promoter. One of ordinaryskill in the art is familiar with such antisense technology. Any nucleicacid sequence may be negatively regulated in this manner. Preferabletarget nucleic acid sequences contain a low content of essential aminoacids, yet are expressed at relatively high levels in particulartissues. For example, β-conglycinin and glycinin are expressedabundantly in seeds, but are nutritionally deficient with respect toessential amino acids. This antisense approach may also be used toeffectively remove other undesirable proteins, such as antifeedants(e.g., lectins), albumin, and allergens, from plant-derived foodstuffs,or to down-regulate catabolic enzymes involved in degradation of desiredcompounds such as essential amino acids.

Selectable Markers

A vector or construct may also include a selectable marker. Selectablemarkers may also be used to select for plants or plant cells thatcontain the exogenous genetic material. Examples of such include, butare not limited to: a neo gene (Potrykus et al., 1985), which codes forkanamycin resistance and can be selected for using kanamycin, RptII,G418, hpt, etc.; a bar gene which codes for bialaphos resistance; amutant EPSP synthase gene (Hinchee et al., 1988; Reynaerts et al.,1988); aadA (Jones et al., 1987) which encodes glyphosate resistance; anitrilase gene which confers resistance to bromoxynil (Stalker et al.,1988); a mutant acetolactate synthase gene (ALS) which confersimidazolinone or sulphonylurea resistance (European Patent Application154,204 (1985)), ALS (D'Halluin et al., 1992), and a methotrexateresistant DHFR gene (Thillet et al., 1988). The selectable marker ispreferably GUS, green fluorescent protein (GFP), neomycinphosphotransferase II (nptII), luciferase (LUX), an antibioticresistance coding sequence, or an herbicide (e.g., glyphosate)resistance coding sequence. The selectable marker is most preferably akanamycin, hygromycin, or herbicide resistance marker.

A vector or construct may also include a screenable marker. Screenablemarkers may be used to monitor expression. Exemplary screenable markersinclude: a β-glucuronidase or uidA gene (GUS) which encodes an enzymefor which various chromogenic substrates are known (Jefferson, 1987); anR-locus gene, which encodes a product that regulates the production ofanthocyanin pigments (red color) in plant tissues (Dellaporta et al.,1988); a β-lactamase gene (Sutcliffe et al., 1978), a gene which encodesan enzyme for which various chromogenic substrates are known (e.g.,PADAC, a chromogenic cephalosporin); a luciferase gene (Ow et al.,1986); a xylE gene (Zukowsky et al., 1983) which encodes a catecholdioxygenase that can convert chromogenic catechols; an α-amylase gene(Ikatu et al., 1990); a tyrosinase gene (Katz et al., 1983) whichencodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinonewhich in turn condenses to melanin; an α-galactosidase, which will turna chromogenic α-galactose substrate.

Included within the term or phrase “selectable or screenable markergenes” are also genes which encode a secretable marker whose secretioncan be detected as a means of identifying or selecting for transformedcells. Examples include markers which encode a secretable antigen thatcan be identified by antibody interaction, or even secretable enzymeswhich can be detected catalytically. Secretable proteins fall into anumber of classes, including small, diffusible proteins which aredetectable, (e.g., by ELISA), small active enzymes which are detectablein extracellular solution (e.g. α-amylase, β-lactamase, phosphinothricintransferase), or proteins which are inserted or trapped in the cell wall(such as proteins which include a leader sequence such as that found inthe expression unit of extension or tobacco PR-S). Other possibleselectable and/or screenable marker genes will be apparent to those ofskill in the art.

Other Elements in the Recombinant Vector

Various cis-acting untranslated 5′ and 3′ regulatory sequences may beincluded in the recombinant nucleic acid vector. Any such regulatorysequences may be provided in a recombinant vector with other regulatorysequences. Such combinations can be designed or modified to producedesirable regulatory features.

A 3′ non-translated region typically provides a transcriptionaltermination signal, and a polyadenylation signal which functions inplants to cause the addition of adenylate nucleotides to the 3′ end ofthe mRNA. These may be obtained from the 3′ regions of the nopalinesynthase (nos) coding sequence, a soybean 7Sα′ storage protein codingsequence, the arcelin-5 coding sequence, the albumin coding sequence,and the pea ssRUBISCO E9 coding sequence. Typically, nucleic acidsequences located a few hundred base pairs downstream of thepolyadenylation site serve to terminate transcription. These regions arerequired for efficient polyadenylation of transcribed mRNA.

Translational enhancers may also be incorporated as part of therecombinant vector. Thus the recombinant vector may preferably containone or more 5′ non-translated leader sequences which serve to enhanceexpression of the nucleic acid sequence. Such enhancer sequences may bedesirable to increase or alter the translational efficiency of theresultant mRNA. Preferred 5′ nucleic acid sequences include dSSU 5′,PetHSP70 5′, and GmHSP17.9 5′(U.S. Pat. No. 5,362,865).

The recombinant vector may further comprise a nucleic acid sequenceencoding a transit peptide. This peptide may be useful for directing aprotein to the extracellular space, a plastid, or to some othercompartment inside or outside of the cell. (see, e.g., EP 0 218 571,U.S. Pat. Nos. 4,940,835; 5,88,624; 5,610,041; 5,618,988; and6,107,060).

The structural nucleic acid sequence in the recombinant vector maycomprise introns. The introns may be heterologous with respect to thestructural nucleic acid sequence. Preferred introns include the riceactin intron and the corn HSP70 intron.

Fusion Proteins

Any of the above described structural nucleic acid sequences, andmodified forms thereof, may be linked with additional nucleic acidsequences to encode fusion proteins. The additional nucleic acidsequence preferably encodes at least 1 amino acid, peptide, or protein.Many possible fusion combinations exist.

For instance, the fusion protein may provide a “tagged” epitope tofacilitate detection of the fusion protein, such as GST, GFP, FLAG, orpolyHIS. Such fusions preferably encode between 1 and 50 amino acids,more preferably between 5 and 30 additional amino acids, and even morepreferably between 5 and 20 amino acids.

Alternatively, the fusion may provide regulatory, enzymatic, cellsignaling, or intercellular transport functions. For example, a sequenceencoding a plastid transit peptide may be added to direct a fusionprotein to the chloroplasts within seeds. Such fusion partnerspreferably encode between 1 and 1000 additional amino acids, morepreferably between 5 and 500 additional amino acids, and even morepreferably between 10 and 250 amino acids.

Sequence Analysis

In the present invention, sequence similarity or identity is preferablydetermined using the “Best Fit” or “Gap” programs of the SequenceAnalysis Software Package™ (Version 10; Genetics Computer Group, Inc.,University of Wisconsin Biotechnology Center, Madison, Wis.). “Gap”utilizes the algorithm of Needleman and Wunsch (1970) to find thealignment of 2 sequences that maximizes the number of matches andminimizes the number of gaps. “BestFit” performs an optimal alignment ofthe best segment of similarity between two sequences. Optimal alignmentsare found by inserting gaps to maximize the number of matches using thelocal homology algorithm of Smith and Waterman (Smith and Waterman,1981; Smith et al., 1983).

The Sequence Analysis Software Package described above contains a numberof other useful sequence analysis tools for identifying homologues ofthe presently disclosed nucleotide and amino acid sequences. Forexample, the “BLAST” program searches for sequences similar to a querysequence (either peptide or nucleic acid) in a specified database (e.g.,sequence databases maintained at the National Center for BiotechnologyInformation (NCBI) in Bethesda, Md.); “FastA” (Lipman and Pearson, 1985;see, also, Pearson and Lipman, 1988; Pearson, 1990) performs a Pearsonand Lipman search for similarity between a query sequence and a group ofsequences of the same type (nucleic acid or protein); “TfastA” performsa Pearson and Lipman search for similarity between a protein querysequence and any group of nucleotide sequences (it translates thenucleotide sequences in all 6 reading frames before performing thecomparison); “FastX” performs a Pearson and Lipman search for similaritybetween a nucleotide query sequence and a group of protein sequences,taking frameshifts into account. “TfastX” performs a Pearson and Lipmansearch for similarity between a protein query sequence and any group ofnucleotide sequences, taking frameshifts into account (it translatesboth strands of the nucleic acid sequence before performing thecomparison).

Probes and Primers

Short nucleic acid sequences having the ability to specificallyhybridize to complementary nucleic acid sequences may be produced andutilized in the present invention. Such short nucleic acid molecules maybe used as probes to identify the presence of a complementary nucleicacid sequence in a given sample. Thus, by constructing a nucleic acidprobe that is complementary to a small portion of a particular nucleicacid sequence, the presence of that nucleic acid sequence may bedetected and assessed.

Alternatively, the short nucleic acid sequences may be used asoligonucleotide primers to amplify or mutate a complementary nucleicacid sequence using PCR technology. These primers may also facilitatethe amplification of related complementary nucleic acid sequences (e.g.,related nucleic acid sequences from other species).

Short nucleic acid sequences may be used as primers and specifically asPCR primers. A PCR probe is a nucleic acid molecule capable ofinitiating a polymerase activity while in a double-stranded structurewith another nucleic acid. Various methods for determining the structureof PCR primers and PCR techniques exist in the art. Computer generatedsearches using programs such as Primer3(www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi), STSPipeline(www-genome.wi.mit.edu/cgi-bin/www.STS_Pipeline), or GeneUp (Pesole etal., 1998), for example, can be used to identify potential PCR primers.

Any of the nucleic acid sequences disclosed herein may be used as aprimer or probe. Use of these probes or primers may greatly facilitatethe identification of transgenic plants which contain the presentlydisclosed promoters and structural nucleic acid sequences. Such probesor primers may also be used to screen cDNA or genomic libraries foradditional nucleic acid sequences related to or sharing homology withthe presently disclosed promoters and structural nucleic acid sequences.

A primer or probe is generally complementary to a portion of a nucleicacid sequence that is to be identified, amplified, or mutated and ofsufficient length to form a stable and sequence-specific duplex moleculewith its complement. The primer or probe preferably is about 10 to about200 nucleotides long, more preferably is about 10 to about 100nucleotides long, even more preferably is about 10 to about 50nucleotides long, and most preferably is about 14 to about 30nucleotides long.

The primer or probe may, for example without limitation, be prepared bydirect chemical synthesis, by PCR (U.S. Pat. Nos. 4,683,195 and4,683,202), or by excising the nucleic acid specific fragment from alarger nucleic acid molecule.

Transgenic Plants and Transformed Plant Host Cells

The present invention is also directed to transgenic plants andtransformed host cells which comprise a promoter operably linked to aheterologous structural nucleic acid sequence. Other nucleic acidsequences may also be introduced into the plant or host cell along withthe promoter and structural nucleic acid sequence. These other sequencesmay include 3′ transcriptional terminators, 3′ polyadenylation signals,other untranslated nucleic acid sequences, transit or targetingsequences, selectable markers, enhancers, and operators. Preferrednucleic acid sequences of the present invention, including recombinantvectors, structural nucleic acid sequences, promoters, and otherregulatory elements, are described above.

In a preferred embodiment, the transgenic plants and transformed hostcells comprise a USP promoter from Vicia faba. In a most preferredembodiment, the transgenic plants and transformed host cells compriseany nucleic acid molecule of the present invention as described herein,including a nucleic acid sequence selected from the group consisting ofSEQ ID NOS: 1, 2, 3, 4, 9, 10, and 11, and complements thereof.

In a particularly preferred embodiment, the transgenic plant of thepresent invention is a soybean plant. In a preferred embodiment, asoybean plant of the present invention comprises one or more introducednucleic acid molecules of the present invention. In a preferredembodiment, a transformed soybean plant of the present inventioncomprises a nucleic acid molecule selected from the group consisting ofSEQ ID NOS: 1, 2, 3, 4, 5, 9, 10, and 11. In a preferred embodiment atransformed soybean plant of the present invention comprises a nucleicacid molecule comprising SEQ ID NO: 4. In a preferred embodiment atransformed soybean plant of the present invention comprises a nucleicacid molecule comprising SEQ ID NO: 5.

In some embodiments of the present invention, one or more components ofa plant, cell, or organism are compared to a plant, cell, or organismhaving a “similar genetic background.” In a preferred aspect, a “similargenetic background” is a background where the organisms being comparedshare about 50% or greater of their nuclear genetic material. In a morepreferred aspect a similar genetic background is a background where theorganisms being compared share about 75% or greater, even morepreferably about 90% or greater of their nuclear genetic material. Inanother even more preferable aspect, a similar genetic background is abackground where the organisms being compared are plants, and the plantsare isogenic except for any genetic material originally introduced usingplant transformation techniques.

Means for preparing such recombinant vectors are well known in the art.For example, methods for making recombinant vectors particularly suitedto plant transformation are described in U.S. Pat. Nos. 4,971,908;4,940,835; 4,769,061; and 4,757,011. These vectors have also beenreviewed (Rodriguez et al., 1988; Glick et al., 1993).

Typical vectors useful for expression of nucleic acids in cells andhigher plants are well known in the art and include vectors derived fromthe tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens (Rogers etal, 1987). Other recombinant vectors useful for plant transformationhave also been described (Fromm et al., 1985). Elements of suchrecombinant vectors include, without limitation, those discussed above.

A transformed host cell may generally be any cell that is compatiblewith the present invention. A transformed host plant or cell can be orderived from a monocotyledonous plant or a dicotyledonous plantincluding, but not limited to canola, crambe, maize, mustard, castorbean, sesame, cottonseed, linseed, soybean, Arabidopsis phaseolus,peanut, alfalfa, wheat, rice, oat, sorghum, rapeseed, rye, tritordeum,millet, fescue, perennial ryegrass, sugarcane, cranberry, papaya,banana, safflower, oil palms, flax, muskmelon, apple, cucumber,dendrobium, gladiolus, chrysanthemum, liliacea, cotton, eucalyptus,sunflower, Brassica campestris, Brassica napus, turfgrass, sugarbeet,coffee, and dioscorea (Christou, In: Particle Bombardment for GeneticEngineering of Plants, Biotechnology Intelligence Unit, Academic Press,San Diego, Calif. (1996)), with canola, maize, Brassica campestris,Brassica napus, rapeseed, soybean, safflower, wheat, rice, and sunflowerpreferred, and canola, rapeseed, maize, Brassica campestris, Brassicanapus, soybean, sunflower, safflower, oil palms, and peanut morepreferred. In a particularly preferred embodiment, the plant or cell isor derived from canola. In another particularly preferred embodiment,the plant or cell is or derived from Brassica napus. In anotherparticularly preferred embodiment, the plant or cell is or derived fromsoybean.

The soybean cell or plant is preferably an elite soybean cell line. An“elite line” is any line that has resulted from breeding and selectionfor superior agronomic performance. Examples of elite lines are linesthat are commercially available to farmers or soybean breeders such asHARTZ™ variety H4994, HARTZ™ variety H5218, HARTZ™ variety H5350, HARTZ™variety H5545, HARTZ™ variety H5050, HARTZ™ variety H5454, HARTZ™variety H5233, HARTZ™ variety H5488, HARTZ™ variety HLA572, HARTZ™variety H6200, HARTZ™ variety H6104, HARTZ™ variety H6255, HARTZ™variety H6586, HARTZ™ variety H6191, HARTZ™ variety H7440, HARTZ™variety H4452 Roundup Ready™, HARTZ™ variety H4994 Roundup Ready™,HARTZ™ variety H4988 Roundup Ready™, HARTZ™ variety H5000 RoundupReady™, HARTZ™ variety H5147 Roundup Ready™, HARTZ™ variety H5247Roundup Ready™, HARTZ™ variety H5350 Roundup Ready™, HARTZ™ varietyH5545 Roundup Ready™, HARTZ™ variety H5855 Roundup Ready™, HARTZ™variety H5088 Roundup Ready™, HARTZ™ variety H5164 Roundup Ready™,HARTZ™ variety H5361 Roundup Ready™, HARTZ™ variety H5566 RoundupReady™, HARTZ™ variety H5181 Roundup Ready™, HARTZ™ variety H5889Roundup Ready™, HARTZ™ variety H5999 Roundup Ready™, HARTZ™ varietyH6013 Roundup Ready™, HARTZ™ variety H6255 Roundup Ready™, HARTZ™variety H6454 Roundup Ready™, HARTZ™ variety H6686 Roundup Ready™,HARTZ™ variety H7152 Roundup Ready™, HARTZ™ variety H7550 RoundupReady™, HARTZ™ variety H8001 Roundup Ready™ (HARTZ SEED, Stuttgart,Ark.); A0868, AGO901, A1553, A1900, AG1901, A1923, A2069, AG2101,AG2201, A2247, AG2301, A2304, A2396, AG2401, AG2501, A2506, A2553,AG2701, AG2702, A2704, A2833, A2869, AG2901, AG2902, AG3001, AG3002,A3204, A3237, A3244, AG3301, AG3302, A3404, A3469, AG3502, A3559,AG3601, AG3701, AG3704, AG3750, A3834, AG3901, A3904, A4045 AG4301,A4341, AG4401, AG4501, AG4601, AG4602, A4604, AG4702, AG4901, A4922,AG5401, A5547, AG5602, A5704, AG5801, AG5901, A5944, A5959, AG6101,QR4459, and QP4544 (Asgrow Seeds, Des Moines, Iowa); DeKalb varietyCX445 (DeKalb, Ill.).

The present invention is also directed to a method of producingtransformed plants which comprise, in a 5′ to 3′ orientation, a promoteroperably linked to a heterologous structural nucleic acid sequence.Other sequences may also be introduced into plants along with thepromoter and structural nucleic acid sequence. These other sequences mayinclude 3′ transcriptional terminators, 3′ polyadenylation signals,other untranslated sequences, transit or targeting sequences, selectablemarkers, enhancers, and operators. Preferred recombinant vectors,structural nucleic acid sequences, promoters, and other regulatoryelements including, without limitation, those described herein.

The method generally comprises the steps of selecting a suitable plant,transforming the plant with a recombinant vector, and obtaining thetransformed host cell.

There are many methods for introducing nucleic acids into plants.Suitable methods include bacterial infection (e.g., Agrobacterium),binary bacterial artificial chromosome vectors, direct delivery ofnucleic acids (e.g., via PEG-mediated transformation,desiccation/inhibition-mediated nucleic acid uptake, electroporation,agitation with silicon carbide fibers, and acceleration of nucleic acidcoated particles, etc. (reviewed in Potrykus et al., 1991)).

Technology for introduction of nucleic acids into cells is well known tothose of skill in the art. Methods can generally be classified into fourcategories: (1) chemical methods (Graham and van der Eb, 1973; Zatloukalet al., 1992); (2) physical methods such as microinjection (Capecchi,1980), electroporation (Wong and Neumann, 1982; Fromm et al., 1985; U.S.Pat. No. 5,384,253), and particle acceleration (Johnston and Tang, 1994;Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu et al., 1993;Eglitis and Anderson, 1988); and (4) receptor-mediated mechanisms(Curiel et al., 1992; Wagner et al., 1992). Alternatively, nucleic acidscan be directly introduced into pollen by directly injecting a plant'sreproductive organs (Zhou et al., 1983; Hess, 1987; Luo et al, 1988;Pena et al., 1987). In another aspect nucleic acids may also be injectedinto immature embryos (Neuhaus et al., 1987).

Regeneration, development, and cultivation of plants from transformedplant protoplast or explants is taught in the art (Weissbach andWeissbach, 1988; Horsch et al., 1985). Transformants are generallycultured in the presence of a selective media which selects for thesuccessfully transformed cells and induces the regeneration of plantshoots (Fraley et al., 1983). Such shoots are typically obtained within2 to 4 months.

Shoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Many of the shoots will develop roots. These are thentransplanted to soil or other media to allow the continued developmentof roots. The method, as outlined, will generally vary depending on theparticular plant employed.

Preferably, the regenerated transgenic plants are self-pollinated toprovide homozygous transgenic plants. Alternatively, pollen obtainedfrom the regenerated transgenic plants may be crossed withnon-transgenic plants, preferably inbred lines of agronomicallyimportant species. Conversely, pollen from non-transgenic plants may beused to pollinate the regenerated transgenic plants.

A transgenic plant may pass along the nucleic acid sequence encoding theenhanced gene expression to its progeny. The transgenic plant ispreferably homozygous for the nucleic acid encoding the enhanced geneexpression and transmits that sequence to all of its offspring upon as aresult of sexual reproduction. Progeny may be grown from seeds producedby the transgenic plant. These additional plants may then beself-pollinated to generate a true breeding line of plants.

The progeny from these plants are evaluated, among other things, forgene expression. The gene expression may be detected by several commonmethods such as western blotting, northern blotting,immunoprecipitation, and ELISA.

Plants or agents of the present invention can be utilized in methods,for example without limitation, to obtain a seed that expresses astructural nucleic acid molecule in that seed, to obtain a seed enhancedin a product of a structural gene, to obtain meal enhanced in a productof a structural gene, to obtain feedstock enhanced in a product of astructural gene, and to obtain oil enhanced in a product of a structuralgene

Plants utilized in such methods may be processed. A plant or plant partmay be separated or isolated from other plant parts. A preferred plantpart for this purpose is a seed. It is understood that even afterseparation or isolation from other plant parts, the isolated orseparated plant part may be contaminated with other plant parts. In apreferred aspect, the separated plant part is greater than about 50%(w/w) of the separated material, more preferably, greater than about 75%(w/w) of the separated material, and even more preferably greater thanabout 90% (w/w) of the separated material. Plants or plant parts of thepresent invention generated by such methods may be processed intoproducts using known techniques. Preferred products are-meal, feedstock,and oil.

Feed, Meal, Protein, and Oil Preparations

Any of the plants or parts thereof of the present invention may beprocessed to produce a feed, meal, protein, or oil preparation. Aparticularly preferred plant part for this purpose is a seed. In apreferred embodiment the feed, meal, protein, or oil preparation isdesigned for ruminant animals. Methods to produce feed, meal, protein,and oil preparations are known in the art. See, for example, U.S. Pat.Nos. 4,957,748; 5,100,679; 5,219,596; 5,936,069; 6,005,076; 6,146,669;and 6,156,227. In a preferred embodiment, the protein preparation is ahigh protein preparation. Such a high protein preparation preferably hasa protein content of greater than about 5% w/v, more preferably about10% w/v, and even more preferably about 15% w/v. In a preferred oilpreparation, the oil preparation is a high oil preparation with an oilcontent derived from a plant or part thereof of the present invention ofgreater than about 5% w/v, more preferably greater than about 10% w/v,and even more preferably greater than about 15% w/v. In a preferredembodiment the oil preparation is a liquid and of a volume greater than1, 5, 10, or 50 liters. The present invention provides for oil producedfrom plants of the present invention or generated by a method of thepresent invention. Such oil may be a minor or major component of anyresultant product. Moreover, such oil may be blended with other oils. Ina preferred embodiment, the oil produced from plants of the presentinvention or generated by a method of the present invention constitutesgreater than about 0.5%, 1%, 5%, 10%, 25%, 50%, 75%, or about 90% byvolume or weight of the oil component of any product. In anotherembodiment, the oil preparation may be blended and can constitutegreater than about 10%, 25%, 35%, 50%, or about 75% of the blend byvolume. Oil produced from a plant of the present invention can beadmixed with one or more organic solvents or petroleum distillates.

In a further embodiment, meal of the present invention may be blendedwith other meals. In a preferred embodiment, the meal produced fromplants of the present invention or generated by a method of the presentinvention constitutes greater than about 0.5%, 1%, 5%, 10%, 25%, 50%,75%, or about 90% by volume or weight of the meal component of anyproduct. In another embodiment, the meal preparation may be blended andcan constitute greater than about 10%, 25%, 35%, 50%, or about 75% ofthe blend by volume.

Seed containers

Seeds of the plants may be placed in a container. As used herein, acontainer is any object capable of holding such seeds. A containerpreferably contains greater than about 500, 1,000, 5,000, or about25,000 seeds where at least about 10%, 25%, 50%, 75%, or about 100% ofthe seeds are derived from a plant of the present invention.

Breeding Programs

Plants of the present invention can be part of or generated from abreeding program. The choice of breeding method depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc.). Selected, non-limiting approaches, forbreeding the plants of the present invention are set forth below. Abreeding program can be enhanced using marker assisted selection of theprogeny of any cross. It is further understood that any commercial andnon-commercial cultivars can be utilized in a breeding program. Factorssuch as, for example, emergence vigor, vegetative vigor, stresstolerance, disease resistance, branching, flowering, seed set, seedsize, seed density, standability, and threshability, etc. will generallydictate the choice.

For highly heritable traits, a choice of superior individual plantsevaluated at a single location will be effective, whereas for traitswith low heritability, selection should be based on mean values obtainedfrom replicated evaluations of families of related plants. Popularselection methods commonly include pedigree selection, modified pedigreeselection, mass selection, and recurrent selection. In a preferredembodiment a backcross or recurrent breeding program is undertaken.

The complexity of inheritance influences choice of the breeding method.Backcross breeding can be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Breeding lines can be tested and compared to appropriate standards inenvironments representative of the commercial target area(s) for two ormore generations. The best lines are candidates for new commercialcultivars; those still deficient in traits may be used as parents toproduce new populations for further selection.

One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations can provide a better estimate of its genetic worth. Abreeder can select and cross two or more parental lines, followed byrepeated selfing and selection, producing many new genetic combinations.

The development of new cultivars requires the development and selectionof varieties, the crossing of these varieties and the selection ofsuperior hybrid crosses. The hybrid seed can be produced by manualcrosses between selected male-fertile parents or by using male sterilitysystems. Hybrids are selected for certain single gene traits such as podcolor, flower color, seed yield, pubescence color, or herbicideresistance, which indicate that the seed is truly a hybrid. Additionaldata on parental lines, as well as the phenotype of the hybrid,influence the breeder's decision whether to continue with the specifichybrid cross.

Pedigree breeding and recurrent selection breeding methods can be usedto develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. New cultivarscan be evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents who possess favorable, complementarytraits are crossed to produce an F₁. An F₂ population is produced byselfing one or several F₁'s. Selection of the best individuals from thebest families is carried out. Replicated testing of families can beginin the F₄ generation to improve the effectiveness of selection fortraits with low heritability. At an advanced stage of inbreeding (i.e.,F₆ and F₇), the best lines or mixtures of phenotypically similar linesare tested for potential release as new cultivars.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line, which is the recurrent parent. The source of the traitto be transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting parent is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, breeders commonly harvest one or more podsfrom each plant in a population and thresh them together to form a bulk.Part of the bulk is used to plant the next generation and part is put inreserve. The procedure has been referred to as modified single-seeddescent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seeds of apopulation each generation of inbreeding.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Fehr, Principles of Cultivar Development, Vol. 1, pp. 2–3(1987)).

A transgenic plant of the present invention may also be reproduced usingapomixis. Apomixis is a genetically controlled method of reproduction inplants where the embryo is formed without union of an egg and a sperm.There are three basic types of apomictic reproduction: 1) apospory wherethe embryo develops from a chromosomally unreduced egg in an embryo sacderived from the nucleus, 2) diplospory where the embryo develops froman unreduced egg in an embryo sac derived from the megaspore mothercell, and 3) adventitious embryony where the embryo develops directlyfrom a somatic cell. In most forms of apomixis, pseudogamy, orfertilization of the polar nuclei to produce endosperm is necessary forseed viability. In apospory, a nurse cultivar can be used as a pollensource for endosperm formation in seeds. The nurse cultivar does notaffect the genetics of the aposporous apomictic cultivar since theunreduced egg of the cultivar develops parthenogenetically, but makespossible endosperm production. Apomixis is economically important,especially in transgenic plants, because it causes any genotype, nomatter how heterozygous, to breed true. Thus, with apomicticreproduction, heterozygous transgenic plants can maintain their geneticfidelity throughout repeated life cycles. Methods for the production ofapomictic plants are known in the art. See, U.S. Pat. No. 5,811,636.

Other Organisms

A nucleic acid of the present invention may be introduced into any cellor organism such as a mammalian cell, mammal, fish cell, fish, birdcell, bird, algae cell, algae, fungal cell, fungi, or bacterial cell.Preferred host and transformants include: fungal cells such asAspergillus, yeasts, mammals, particularly bovine and porcine, insects,bacteria, and algae. Preferred bacteria are E. coli and Agrobacteriumtumefaciens.

Methods to transform such cells or organisms are known in the art (EP 0238 023; Yelton et al, 1984; Malardier et al., 1989; Becker andGuarente; Ito et al., 1983; Hinnen et al., 1978; and Bennett and LaSure,1991). Methods to produce proteins from such organisms are also known(Kudla et al., 1990; Jarai and Buxton, 1994; Verdier, 1990; MacKenzie etal., 1993; Hartl et al., 1994; Bergeron et al., 1994; Demolder et al,1994; Craig, 1993; Gething and Sambrook, 1992; Puig and Gilbert, 1994;Wang and Tsou, 1993; Robinson et al., 1994; Enderlin and Ogrydziak,1994; Fuller et al., 1989; Julius et al., 1984; and Julius et al.,1983).

EXAMPLES

The following examples are provided and should not be interpreted in anyway to limit the scope of the present invention.

Example 1 Generation of Clones of USP Promoters from Vicia faba

USP promoters are obtained from Vicia faba genomic DNA via PCRamplification (Expand High Fidelity PRC System, Cat# 1 732 641, RocheMolecular Biochemicals, Indianapolis, Ind.) using primers designedaccording to the published sequence (GenBank Accession X56240). Primersused for amplification of the USP promoters are:

5′-AAACTGCAGCAAATTTACACATTG-3′; and (SEQ ID NO: 6)5′-AAACCATGGTTGACTGGCTATG-3′. (SEQ ID NO: 7)The isolated amplification products are then subcloned into the vectorpMON13773 (FIG. 1) producing clones pMON58101 (USP99) (FIG. 2),pMON58102 (USP91) (FIG. 3), and pMON58106 (USP88) (FIG. 4).

Example 2 Generation of a Chimeric eUSP88 Promoter

A PCR reaction is performed using pMON58106 as a template (Expand HighFidelity PRC System, Catalog number 1 732 641, Roche MolecularBiochemicals, Indianapolis, Ind.). The following primers are used foramplification:

5′-AAACTGCAGCAAATTTACACATTG-3′; and (SEQ ID NO: 6)5′-AAACTGCAGGACTACATGCATAAC-3′. (SEQ ID NO: 8)The amplified promoter fragments are digested using Pst I restrictionenzyme and are ligated to pMON58106 DNA which are linearized by Pst Idigestion and treated with CIP alkaline phosphatase. A plasmid with thedesired orientation is selected and designated as pMON581 10 (eUSP88)(FIG. 5).

Example 3 Evaluation of USP Promoters Using Soybean Cotyledon TransientTransformation

Seeds from soybean plants (Asgrow A3244) are harvested 25–28 days afterflowering and osmotically treated overnight at 25° C. in dark onGAMBORG's medium (G5893, Sigma Company, St. Louis, Mo.) supplementedwith of 50 mM glutamine, 111 mM maltose, 125 mM raffinose, 125 mMmannitol and 3 g/l purified agar, pH 5.6. The resulting 125 cotyledonsare cut in half and bombarded with purified supercoiled DNA of pMON13773(7Sα′), pMON58101 (USP99), pMON58102 (USP91), pMON58106 (USP88), andpMON58110 (Minimum 35S) using particle gun technology (Maliga et al.,1995, “Methods in Plant Molecular Biology, A Laboratory Course Manual,”Cold Spring Harbor Laboratory Press, page 47). A separate e35S drivenluciferase construct is included in a 1:1 molar ratio with each of thepromoter constructs as a low expression control. Bombarded tissues areincubated for 48 hours at 25° C.

Proteins are extracted from six bombarded soybean cotyledons using 1 mlextraction buffer containing 0.1 M potassium phosphate (pH 7.8), 10 mMDTT, 1 mM EDTA, 5% glycerol, and proteinase inhibitor (1 tablet/50ml,Roche Molecular Biochemicals, Catalog number 1 697 498, Indianapolis,Ind.). A 100 μl aliquot of the protein extract is used for Luciferaseassay following a “Steady-Glo” procedure by Promega (Catalog numberE2510, Promega Corporation, Madison, Wis.). A 50 μl aliquot of theprotein extract is used for a standard GUS assay protocol with minormodifications (Maliga et al., 1995, “Methods in Plant Molecular Biology,A Laboratory Course Manual”, Cold Spring Harbor Laboratory Press, page29). Each sample is assayed twice and the average value is used for dataanalysis. GUS activity is normalized using luciferase activity and therelative promoter strength is expressed by setting the benchmarkpromoter 7Sα′ (pMON13773) (FIG. 1) arbitrarily at 100%. The experimentis repeated independently three times. The results (FIG. 14) indicatethat the USP promoters significantly increase expression of GUS whencompared to the 7Saα′ promoter, a benchmark promoter traditionally usedfor high level expression in soybean seeds. The minimal promoter/GUSconstruct (pMON58100), which is a low expression control using a 35minimum promoter driving GUS) is expressed at a level of approximately20% of that of a 7Sα′ construct (pMON13773) (FIG. 1).

Example 4 Production of Transgenic Soybean Plants Containing USPPromoters

To test the strength of USP promoters in transgenic soybean, pMON55526(Arc5/GUS/NOS), pMON55542 (T-Arc5/GUS/NOS), pMON63605 (USP91/GUS/NOS),pMON58107 (USP88/GUS/NOS), pMON63604 (USP99/GUS/NOS), and pMON58113(eUSP88/GUS/NOS) are constructed by following standard molecular cloningprotocols with minor modification (Sambrook et al., Molecular Cloning: Alaboratory manual, 1989, Cold Spring Harbor Laboratory Press; Maliga etal., Methods in Plant Molecular Biology: A laboratory course manual,1995, Cold Spring Harbor Laboratory Press). An expression cassetteconsisting of an FMV promoter, transit peptide sequence, CP4 coding geneand E9 3′ UTR is included as selectable marker in all transformationvectors.

For the particle bombardment transformation method, soybean seeds(Asgrow A3244, A4922) are germinated overnight (18–24 hours) and themeristem explants are excised. The primary leaves are removed to exposethe meristems. Prepared explants are stored for up to two days at 4° C.in the dark in OR media (see, U.S. Pat. No. 5,914,451 for description ofOR media) and at 15° C. in the dark for one day. Immediately prior tobombardment, the prepared explants are placed in targeting media (see,U.S. Pat. No. 5,914,451 for description of targeting media) with themeristems positioned perpendicular to the direction of the particledelivery. DNA containing, pMON55526, pMON58107, or pMON55542 isprecipitated onto microscopic gold particles with CaCl₂ and spermidineand subsequently resuspended in ethanol. The suspension is coated onto amylar sheet which is then placed onto the electric discharge device. Theparticles are accelerated into the plant tissue by electric discharge atapproximately 60% capacitance. Typically, targets are bombarded once.Following bombardment, explants are placed in selection media (WPM +75μM glyphosate, see, U.S. Pat. No. 5,914,451 for description) for 5–7weeks to allow selection and growth of transgenic shoots. Phenotypepositive shoots are harvested approximately 5–7 weeks post bombardmentand placed into selective rooting media (BRM +25 microM glyphosate, see,U.S. Pat. No. 5,914,451 for description) for 2–3 weeks. Shoots producingroots are transferred to a greenhouse and potted in soil. Shoots thatremain healthy on selection, but do not produce roots, are transferredto non-selective rooting media (BRM, as above) for an additional twoweeks. Roots from any shoots that produce roots off selection are testedfor expression of the plant selectable marker before they aretransferred to the green house and potted in soil. Plants are maintainedunder standard green house conditions until R1 seed harvest.

For the Agrobacterium transformation method, commercially availablesoybean seeds (Asgrow A3244, A4922) are germinated overnight(approximately 10–12 hours) and the meristem explants are excised. Theprimary leaves may or may not be removed to expose the meristems and theexplants are placed in a wounding vessel. Agrobacterium strain ABIcontaining pMON58113 (FIG. 8), pMON63605, or pMON63604 is grown to logphase. Cells are harvested by centrifugation and resuspended ininoculation media containing inducers. Soybean explants and the inducedAgrobacterium culture are mixed no later than 14 hours from the time ofinitiation of seed germination and wounded using sonication.

Following wounding, explants are incubated in Agrobacterium for a periodof approximately one hour. Following this inoculation step, theAgrobacterium is removed by pipetting and the explants are placed inco-culture for 2–4 days. They are transferred to selection media(WPM+0.075 mM glyphosate+antibiotics to control Agrobacteriumovergrowth) for 5–7 weeks to allow selection and growth of transgenicshoots. Phenotype positive shoots are harvested approximately 5–7 weekspost-bombardment and placed into selective rooting media (BRM +0.025 mMglyphosate) for 2–3 weeks. Shoots producing roots are transferred to thegreenhouse and potted in soil. Shoots that remained healthy onselection, but did not produce roots were transferred to non-selectiverooting media (BRM without glyphosate) for an additional 2 weeks. Theroots from any shoots that produced roots off the selection are testedfor expression of the plant selectable marker glyphosate resistancebefore transferring to the greenhouse and potting in soil. Plants aremaintained under standard greenhouse conditions until R1 seed harvest.

Mature seeds from the selected plants are analyzed for GUS activity. Toassay for GUS activity, eight seeds from each transgenic event (line)are ground individually. About 20 mg ground seed tissue is extractedusing 200 μl extraction buffer containing 0.1 M potassium phosphate (pH7.8), 10 mM DTT, 1 mM EDTA, 5% glycerol, and proteinase inhibitor (1tablet/50 ml, Catalog number 1 697 498, Roche Molecular Biochemicals,Indianapolis, Ind.). The protein content of the extract is determinedusing Bio-Rad Protein Assay (Catalog number 61234A, Bio-RadLaboratories, Hercules, Calif.) and the GUS activity is measured using astandard GUS assay protocol with minor modifications (Maliga et al.,1995, “Methods in Plant Molecular Biology, A Laboratory Course Manual”,Cold Spring Harbor Laboratory Press, p. 29). The GUS activity isnormalized against the protein concentration. Each sample is assayedtwice and the average value is used for data analysis.

An event (line) is rejected if none of the 8 seeds had detectable GUSactivity. If at least one seed of a particular event has detectable GUS,the event is considered a positive transgenic event. The seed samplewith highest GUS activity within the event is chosen as therepresentative of the event because it is more likely to reflect GUSactivity in homozygous seeds. Between 10–20 positive events aretypically identified for each of the constructs. The average of thepositive events are compared to demonstrate the relative strength of thepromoter as shown in FIG. 12. The data show that the USP88 is about 3times stronger than the T-Arc5 promoter, and about 6 times stronger thanthe Arc5 promoter in transgenic soybean seeds (FIG. 12). The result alsoshows that the eUSP88 promoter is about 10 times stronger than theT-Arc5 promoter in transgenic soybean seeds (FIG. 12).

Example 5 Production of Transgenic Soybean Plants Containing ElevatedLevel of Free Tryptophan in Seeds

An Agrobacterium transformation vector pMON58130 (FIG. 13) is created todemonstrate the effectiveness of the USP99 promoter at driving atryptophan-insensitive α-subunit of anthranilate synthase from C28 maize(U.S. Pat. No. 6,118,047) in transgenic soybean. Construction of vectorpMON58130 (FIG. 13) is done by following standard molecular cloningprotocols with minor modification (Sambrook et al., Molecular Cloning: Alaboratory manual, 1989, Cold Spring Harbor Laboratory Press; Maliga etal., Methods in Plant Molecular Biology: A laboratory course manual,1995, Cold Spring Harbor Laboratory Press). An expression cassetteconsisting of an FMV promoter, a transit peptide sequence, a CP4 codinggene, and an E9 3′ UTR is included as a selectable marker in thetransformation vector.

An additional Agrobacterium vector pMON63654 is created to demonstratethe effectiveness of the USP99 promoter at driving a tryptophan feedbackinsensitive mutant (F298W) of anthranilate synthase from Agrobacteriumtumefaciens (U.S. patent application Ser. No. 60/288,904) in transgenicsoybean. Construction of vector pMON63654 (FIG. 21) is done by followingstandard molecular cloning protocols with minor modification (Sambrooket al., Molecular Cloning: A laboratory manual, 1989, Cold Spring HarborLaboratory Press; Maliga et al., Methods in Plant Molecular Biology: Alaboratory course manual, 1995, Cold Spring Harbor Laboratory Press). Anexpression cassette consisting of the FMV promoter with the HSP70 5′UTR, CTP2 and CP4 coding gene and E9 3′ UTR is included as selectablemarker in this vector. In pMON63654 (FIG. 21), the USP99 promoter isligated upstream of a gene consisting of a chloroplast transit peptidesequence CTP 1 and an Agrobacterium anthranilate synthase (F298W)mutant. A NOS 3′ UTR is used to signal transcription termination andpolyadenylation.

For the Agrobacterium transformation method, commercially availablesoybean seeds (Asgrow A3244, A4922) are germinated overnight(approximately 10–12 hours) and the meristem explants are excised. Theprimary leaves may or may not be removed to expose the meristems and theexplants are placed in a wounding vessel. Agrobacterium strain ABIcontaining pMON58130 or pMON63654 is grown to log phase. Cells areharvested by centrifugation and resuspended in inoculation mediacontaining inducers. Soybean explants and the induced Agrobacteriumculture are mixed no later than 14 hours from the time of initiation ofseed germination and wounded using sonication.

Following wounding, explants are incubated in Agrobacterium for a periodof approximately one hour. Following this inoculation step, theAgrobacterium is removed by pipetting and the explants are placed inco-culture for 2–4 days. At this point, they are transferred toselection media (WPM +0.075 mM glyphosate +antibiotics to controlAgrobacterium overgrowth) for 5–7 weeks to allow selection and growth oftransgenic shoots. Phenotype positive shoots are harvested approximately5–7 weeks post-bombardment and placed into selective rooting media (BRM+0.025 mM glyphosate) for 2–3 weeks. Shoots producing roots aretransferred to the greenhouse and potted in soil. Shoots that remainedhealthy on selection, but did not produce roots are transferred tonon-selective rooting media (BRM without glyphosate) for an additional 2weeks. The roots from any shoots that produced roots off the selectionare tested for expression of the plant selectable marker glyphosateresistance before transferring to the greenhouse and potting in soil.Plants are maintained under standard greenhouse conditions until R1 seedharvest.

To assay for free tryptophan, ten mature R1 seeds from each transgenicevent (line) are crushed individually. About 50 mg of crushed materialfrom individual seed is placed in each centrifuge vial and weighed. Onemilliliter of 5% trichloroacetic acid is added to each sample. Thesamples are vortexed, and mixed at room temperature for 15 min. They arethen microcentrifuged for 15 min at 14,000 rpm. Some of the supernatantis then removed, placed in a HPLC vial, and sealed. Extracted samplesare analyzed for free amino acid using Zorbax Eclipse-AAA Columns andthe Agilent 1100 HPLC (Agilent Technical Publication, “Amino AcidAnalysis Using Zorbax Eclipse-AAA Columns and the Agilent 1100 HPLC.”Mar. 17, 2000). Because the R1 seeds of each event most likely consistof a population of segregating seeds, the seed with the highesttryptophan reading among the 10 seeds of each event is chosen as therepresentative of the group for its high probability of beinghomozygous. The data is summarized in Tables 3 and 4 below.

Ten randomly selected non-transgenic Asgrow A3244 seeds are alsoanalyzed and the one with the highest tryptophan level is included inthe table as a negative control. pMON58130-1 through pMON58130-23represent different events generated using vector pMON58130. Comparedwith non-transgenic A3244, most of the transgenic events have high levelTrp accumulation.Elevated Trp accumulation is detected in multipletransgenic events using pMON58130 and pMON63654.

TABLE 3 Tryptophan Concentration in pMON58130 Events Event number Trp(ppm) A3244 306 pMON58130-1 484 pMON58130-2 3104 pMON58130-3 8237pMON58130-4 7734 pMON58130-5 432 pMON58130-6 4540 pMON58130-7 4698pMON58130-8 361 pMON58130-9 344 pMON58130-10 6435 pMON58130-11 5310pMON58130-12 283 pMON58130-13 200 pMON58130-14 90 pMON58130-15 5479pMON58130-16 6316 pMON58130-17 1516 pMON58130-18 3714 pMON58130-19 4480pMON58130-20 636 pMON58130-21 534 pMON58130-22 872 pMON58130-23 4986

TABLE 4 Tryptophan Concentration in pMON63654 Events pMON Event Averageof Max of TRP number Description number TRP (ppm) (ppm) 63654USP99-F298W-AS 27581 10,591 19,630 63654 USP99-F298W-AS 27654 1,80217,796 63654 USP99-F298W-AS 28034 7,186 21,278

Example 6 Generation of Clones of USP Promoters from Vicia faba

Extended sequence USP promoters are obtained from Vicia faba genomic DNAvia PCR amplification (Expand High Fidelity PCR System, Catalog number 1732 641, Roche Molecular Biochemicals, Indianapolis, Ind.) and theUniversal Genome Walker® kit (catalog number K1807-1 BD Biosciences,Palo Alto, Calif.) using a 3′ primer designed according to the publishedsequence (GenBank Accession X56240). 5′ primers are designed accordingto the Genome Walker protocol. Primers used for the first amplificationof the USP promoters are: GATAAAACAGTGAGATGTGCAAACTCC (uspGW-P-down)(SEQ ID NO: 12) and GTAATACGACTCACTATAGGGC (AP1, Adaptor Primer 1,supplied with kit) (SEQ ID NO: 13).

From these primary PCR products the promoters are amplified using nestedprimers CCATGGAGATCTGACTGGCTATGAAGAAATTATAATCG (uspGW-N-down Bgl2Ncol)(SEQ ID NO: 14) and ACTATAGGGCACGCGTGGT (AP2, Nested Adaptor Primer 2)(SEQ ID NO: 15).

One microliter of the PCR fragment elution is used as template for athird round of PCR using uspGW-N-down Bgl2Ncol andCTGCAGGTCGACGGCCCGGGCTGGT (AP6-Pstl/Srfl) (SEQ ID NO: 16) in order toadd convenient 5′ restriction sites to the putative promoter fragments.The isolated amplification products are then subcloned into the vectorpMON8677 producing clones pMON63821. (USP99.5) (FIG. 18), pMON63819(USP95) (FIG. 19), and pMON63820 (USP68) (FIG. 20).

Example 7 Evaluation of USP Promoters Using Soybean Cotyledon TransientTransformation

Seeds from soybean plants (Asgrow A3244) are harvested 25–28 days afterflowering and osmotically treated overnight at 25° C. in dark onGAMBORG's medium (G5893, Sigma Company, St. Louis, Mo.) supplementedwith of 50 mM glutamine, 111 mM maltose, 125 mM raffinose, 125 mMmannitol and 3 g/l purified agar, pH 5.6. The resulting 125 cotyledonsare cut in half and bombarded with purified supercoiled DNA of pMON63819(USP95), pMON63820 (USP68), pMON63621 (USP99.5), and pMON58101 (USP99)using particle gun technology (Maliga et al., 1995, “Methods in PlantMolecular Biology, A Laboratory Course Manual,” Cold Spring HarborLaboratory Press, p. 47). A separate e35S driven luciferase construct isincluded in a 1:1 molar ratio with each of the promoter constructs as aninternal standard for expression. Bombarded tissues are incubated for 48hours at 25° C.

Proteins are extracted from 6 bombarded soybean cotyledons using 1 mlextraction buffer containing 0.1 M potassium phosphate (pH 7.8), 10 mMDTT, 1 mM EDTA, 5% glycerol, and proteinase inhibitor (1 tablet/50ml,Roche Molecular Biochemicals, Catalog number 1 697 498, Indianapolis,Ind.). A 100 μl aliquot of the protein extract is used for luciferaseassay following a “Steady-Glo” procedure by Promega (Catalog number E2510, Promega Corporation Madison, Wis.). A 50 μl aliquot of the proteinextract is used for a standard GUS assay protocol with minormodifications (Maliga et al., 1995, “Methods in Plant Molecular Biology,A Laboratory Course Manual”, Cold Spring Harbor Laboratory Press, p.29). Each sample is assayed twice and the average value is used for dataanalysis. GUS activity is normalized using luciferase activity and therelative promoter strength is expressed by comparing the expression ofpMON5 8101 (USP99) with that of pMON63 819 (USP95), pMON63820 (USP68),pMON63621 (USP99.5) (Table 5). pMON63621 (USP99.5) shows significantlyhigher expression than USP99, pMON63820 (USP68) shows significantlylower expression than USP99, and pMON63819 (USP95) shows similarexpression to that of USP99. All three constructs were confirmed tocontain active promoters in this transient expression system.

TABLE 5 Promoter Activity in a Transient Expression System RelativeStandard Promoter Construct Promoter GUS activity Error Size (bp)pMON58101 USP99 1.59 0.10 682 pMON63819 USP95 1.47 0.21 1464 pMON63820USP68 0.52 0.17 1301 pMON63821 USP99.5 2.81 0.82 1748References

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1. An isolated nucleic acid molecule comprising a promoter operablylinked to a transcribable nucleic acid, wherein the promoter is selectedfrom the group consisting of SEQ ID NO: 2, and SEQ ID NO:
 10. 2. A planttransformed with the nucleic acid molecule of claim
 1. 3. Thetransformed plant of claim 2, wherein said transcribable nucleic acid isa structural nucleic acid.
 4. The transformed plant of claim 3, whereinsaid structural nucleic acid encodes a protein selected from the groupconsisting of a seed storage protein, a fatty acid pathway enzyme, atocopherol biosynthetic enzyme, an amino acid biosynthetic enzyme, asteroid pathway enzyme, and a starch branching enzyme.
 5. Thetransformed plant of claim 3, wherein said structural nucleic acidencodes a protein selected from the group consisting of anthranilatesynthase, tryptophan decarboxylase, threonine deaminase and aspartatekinase.
 6. The transformed plant of claim 4, wherein said structuralnucleic acid encodes a starch branching enzyme.
 7. The transformed plantof claim 3, wherein said structural nucleic acid is oriented to expressan antisense RNA molecule.
 8. The transformed plant of claim 3, whereinsaid transformed plant is selected from the group consisting of canola,crambe, mustard, castor bean, sesame, cottonseed, linseed, maize,soybean, Arabidopsis phaseolus, peanut, alfalfa, wheat, rice, oat,sorghum, rapeseed, rye, tritordeum, millet, fescue, perennial ryegrass,sugarcane, cranberry, papaya, banana, safflower, oil palms, flax,muskmelon, apple, cucumber, dendrobium, gladiolus, chrysanthemum,liliacea, cotton, eucalyptus, sunflower, Brassica campestris, Brassicanapus, turfgrass, sugarbeet, coffee, and dioscorea.
 9. The transformedplant of claim 3, wherein said transformed plant is soybean.
 10. Thetransformed plant of claim 3, wherein said structural nucleic acid isexpressed in an organ specific manner.
 11. The transformed plant ofclaim 10, wherein said structural nucleic acid is expressed in a seed.12. A method of producing a transformed plant comprising transforming aplant with the nucleic acid molecule of claim
 1. 13. A method ofproducing a transformed seed comprising: (a) growing a plant transformedwith the nucleic acid molecule of claim 1 to produce a seed, whereinsaid transcribable nucleic acid is expressed in said seed; and (b)isolating said seed from said transformed plant.
 14. The host celltransformed with the nucleic acid of claim
 1. 15. The host cellaccording to claim 14, wherein said host cell is selected from the groupconsisting of a bacterial cell, an insect cell, a plant cell, and afungal cell.
 16. The host cell according to claim 15, wherein said hostcell is Agrobacterium tumefaciens.
 17. A transgenic seed produced fromthe transformed plant of claim
 2. 18. A soybean plant transformed withthe nucleic acid molecule of claim
 1. 19. The transformed plant of claim18, wherein said transcribing nucleic acid encodes a protein selectedfrom the group consisting of a seed storage protein, a fatty acidpathway enzyme, a tocopherol biosynthetic enzyme, an amino acidbiosynthetic enzyme, a steroid pathway enzyme, and a starch branchingenzyme.
 20. The transformed soybean plant of claim 18, wherein saidtranscribing nucleic acid encodes a protein selected from the groupconsisting of anthranilate synthase, tryptophan decarboxylase, threoninedeaminase and aspartate kinase.
 21. The transformed soybean plant ofclaim 18, wherein said transcribing nucleic acid encodes a starchbranching enzyme.
 22. The transformed soybean plant of claim 18, whereinsaid transcribing nucleic acid is oriented to express an antisense RNAmolecule.
 23. An isolated nucleic acid molecule comprising thecomplementary sequence of SEQ ID NO: 2 or SEQ ID NO:
 10. 24. The nucleicacid molecule of claim 1, wherein the promoter comprises SEQ ID NO: 2.25. The nucleic acid molecule of claim 1, wherein the promoter comprisesSEQ ID NO: 10.